Non-endogenous, constitutively activated known G protein-coupled receptors

ABSTRACT

The invention disclosed in this patent document relates to transmembrane receptors, more particularly to a human G protein-coupled receptor for which the endogenous ligand is known (“known GPCRs”), and most particularly to mutated (non-endogenous) versions of the known GPCRs for use, most preferably in screening assays for the direct identification of candidate compounds as inverse agonists, agonists and partial agonists.

This patent application claims priority from U.S. ProvisionalApplication No. 60/195,747, filed with the U.S. Patent and TrademarkOffice on Apr. 7, 2000, which is incorporated in its entirety byreference.

FIELD OF THE INVENTION

The invention disclosed in this patent document relates to transmembranereceptors, and more particularly to G protein-coupled receptors forwhich the endogenous ligand has been identified (“known GPCR”), andspecifically to known GPCRs that have been altered to establish orenhance constitutive activity of the receptor. Most preferably, thealtered GPCRs are used for the direct identification of candidatecompounds as receptor agonists, inverse agonists or partial agonists foruse as therapeutic agents.

BACKGROUND OF THE INVENTION

Although a number of receptor classes exist in humans, by far the mostabundant and therapeutically relevant is represented by the Gprotein-coupled receptor (GPCR or GPCRs) class. It is estimated thatthere are some 100,000 genes within the human genome, and of these,approximately 2%, or 2,000 genes, are estimated to code for GPCRs.Receptors, including GPCRs, for which the endogenous ligand has beenidentified are referred to as “known” receptors, while receptors forwhich the endogenous ligand has not been identified are referred to as“orphan” receptors. GPCRs represent an important area for thedevelopment of pharmaceutical products: from approximately 20 of the 100known GPCRs, 60% of all prescription pharmaceuticals have beendeveloped.

GPCRs share a common structural motif. (All these receptors have sevensequences of between 22 to 24 hydrophobic amino acids that form sevenalpha helices, each of which spans the membrane (each span is identifiedby number, i.e., transmembrane-1 (TM-1), transmembrane-2 (TM-2), etc.).The transmembrane helices are joined by strands of amino acids betweentransmembrane-2 and transmembrane-3, transmembrane-4 andtransmembrane-5, and transmembrane-6 and transmembrane-7 on theexterior, or “extracellular” side, of the cell membrane (these arereferred to as “extracellular” regions 1, 2 and 3 (EC-1, EC-2 and EC-3),respectively). The transmembrane helices are also joined by strands ofamino acids between transmembrane-1 and transmembrane-2, transmembrane-3and transmembrane-4, and transmembrane-5 and transmembrane-6 on theinterior, or “intracellular” side, of the cell membrane (these arereferred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3),respectively). The “carboxy” (“C”) terminus of the receptor lies in theintracellular space within the cell, and the “amino” (“N”) terminus ofthe receptor lies in the extracellular space outside of the cell.

Generally, when an endogenous ligand binds with the receptor (oftenreferred to as “activation” of the receptor), there is a change in theconformation of the intracellular region that allows for couplingbetween the intracellular region and an intracellular “G-protein.” Ithas been reported that GPCRs are “promiscuous” with respect to Gproteins, i.e., that a GPCR can interact with more than one G protein.See, Kenakin, T., 43 Life Sciences 1095 (1988). Although other Gproteins exist, currently, Gq, Gs, Gi, Gz and Go are G proteins thathave been identified. Endogenous ligand-activated GPCR coupling with theG-protein begins a signaling cascade process (referred to as “signaltransduction”). Under normal conditions, signal transduction ultimatelyresults in cellular activation or cellular inhibition. It is thoughtthat the IC-3 loop as well as the carboxy terminus of the receptorinteract with the G protein.

Under physiological conditions, GPCRs exist in the cell membrane inequilibrium between two different conformations: an “inactive” state andan “active” state. A receptor in an inactive state is unable to link tothe intracellular signaling transduction pathway to produce a biologicalresponse. Changing the receptor conformation to the active state allowslinkage to the transduction pathway (via the G-protein) and produces abiological response.

A receptor may be stabilized in an active state by an endogenous ligandor a compound such as a drug. Recent discoveries, including but notexclusively limited to modifications to the amino acid sequence of thereceptor, provide means other than endogenous ligands or drugs topromote and stabilize the receptor in the active state conformation.These means effectively stabilize the receptor in an active state bysimulating the effect of an endogenous ligand binding to the receptor.Stabilization by such ligand-independent means is termed “constitutivereceptor activation.”

Traditional ligand-dependent screens seek to indirectly identifycompounds that antagonize the action of the ligand on the receptor in aneffort to prevent ligand-induced activation of the receptor. However,such compounds, sometimes referred to as neutral-antagonists, generallywould not be expected to affect the ligand-independent activity, oroveractivity, of the receptor and the subsequent abnormal cellularresponse that can result from this overactivity. This is particularlyrelevant to a growing number of diseases, such as those identified inthe table below, that have been linked to overactive GPCRs, becausetraditional neutral-antagonists will not block the abnormalligand-independent activity of these receptors.

Background Table 1 Disease Overactive GPCR Schizophrenia 5-HT2A, D2Depression 5-HT2A Hyperthyroidism Thyrotropin Hypertension AngiotensinAT1A Asthma Adenosine A1 Melanoma MC-1 Retinitis Pigmentosa Rhodopsinreceptor

SUMMARY OF THE INVENTION

Disclosed herein are non-endogenous versions of endogenous, known GPCRsand uses thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides graphic results of comparative analysis of aco-transfection of non-endogenous TSHR-A6231 (“signal enhancer”) and anendogenous target receptor, in this case GPR24 (“GPR24 wt”), versusnon-endogenous, constitutively activated versions of the target receptorGPR24 (“T255K,” “T255K/T257R,” “24-IC3-SST2,” “C305Y,” “P271L,” “W269C,”“W269F,” “W269L,” “F265I,” I261Q,” and “D140N”) co-transfected withnon-endogenous TSHR-A623I, utilizing an adenylyl cyclase assay. Thisassay involved the addition of TSH and MCH, the endogenous ligands forTSHR and GPR24, respectively.

FIG. 2 provides graphic results of comparative analysis of aco-transfection of non-endogenous signal enhancer TSBR-A623I (with andwithout TSH) and endogenous target receptor GPR5 (“GPR5 wt”), versusnon-endogenous, constitutively activated target receptor GPR5 (“V224K”)co-transfected with non-endogenous TSHR-A623I (with and without TSH),utilizing an adenylyl cyclase assay.

FIGS. 3A-3E provide a diagrammatic representation of the signal measuredcomparing CMV, non-endogenous, constitutively activated GPCRs, utilizing8XCRE-Luc reporter plasmid.

FIG. 4 provides an illustration of IP₃ production from severalnon-endogenous versions of GPR24 as compared with the endogenous versionof this receptor.

FIG. 5 is a graphic representation of the results of a membrane-basedcyclic AMP assay providing comparative results for constitutivesignaling of TSHR-A623K:Fusion Protein and a control vector (pCMV).

DETAILED DESCRIPTION

The scientific literature that has evolved around receptors has adopteda number of terms to refer to ligands having various effects onreceptors. For clarity and consistency, the following definitions willbe used throughout this patent document. To the extent that thesedefinitions conflict with other definitions for these terms, thefollowing definitions shall control:

AGONISTS shall mean materials (e.g., ligands, candidate compounds) thatactivate the intracellular response when they bind to the receptor, orenhance GTP binding to membranes.

AMINO ACID ABBREVIATIONS used herein are set out in Table A:

TABLE A ALANINE ALA A ARGININE ARG R ASPARAGINE ASN N ASPARTIC ACID ASPD CYSTEINE CYS C GLUTAMIC ACID GLU E GLUTAMINE GLN Q GLYCINE GLY GHISTIDINE HIS H ISOLEUCINE ILE I LEUCINE LEU L LYSINE LYS K METHIONINEMET M PHENYLALANINE PHE F PROLINE PRO P SERINE SER S THREONINE THR TTRYPTOPHAN TRP W TYROSINE TYR Y VALINE VAL V

PARTIAL AGONISTS shall mean materials (e.g., ligands, candidatecompounds) that activate the intracellular response when they bind tothe receptor to a lesser degree/extent than do agonists, or enhance GTPbinding to membranes to a lesser degree/extent than do agonists.

ANTAGONIST shall mean materials (e.g., ligands, candidate compounds)that competitively bind to the receptor at the same site as the agonistsbut which do not activate the intracellular response initiated by theactive form of the receptor, and can thereby inhibit the intracellularresponses by agonists or partial agonists. ANTAGONISTS do not diminishthe baseline intracellular response in the absence of an agonist orpartial agonist.

CANDIDATE COMPOUND, in the context of the disclosed invention, shallmean a small molecule (for example, and not limitation, a chemicalcompound) that is amenable to a screening technique.

COMPOSITION means a material comprising at least one component; a“pharmaceutical composition” is an example of a composition.

COMPOUND EFFICACY shall mean a measurement of the ability of a compoundto inhibit or stimulate receptor functionality, as opposed to receptorbinding affinity. Exemplary means of detecting compound efficacy aredisclosed in the Example section of this patent document.

CODON shall mean a grouping of three nucleotides (or equivalents tonucleotides) which generally comprise a nucleoside (adenosine (A),guanosine (G), cytidine (C), uridine (U) and thymidine (T)) coupled to aphosphate group and which, when translated, encodes an amino acid.

CONSTITUTIVELY ACTIVATED RECEPTOR shall mean a receptor subject toconstitutive receptor activation. A constitutively activated receptorcan be endogenous or non-endogenous.

CONSTITUTIVE RECEPTOR ACTIVATION shall mean stabilization of a receptorin the active state by means other than binding of the receptor with itsendogenous ligand or a chemical equivalent thereof.

CONTACT or CONTACTING shall mean bringing at least two moietiestogether, whether in an in vitro system or an in vivo system.

DIRECTLY IDENTIFYING or DIRECTLY IDENTIFIED, in relationship to thephrase “candidate compound”, shall mean the screening of an candidatecompound against a constitutively activated receptor, preferably aconstitutively activated receptor, and most preferably against aconstitutively activated G protein-coupled cell surface receptor, andassessing the compound efficacy of such compound. This phrase is, underno circumstances, to be interpreted or understood to be encompassed byor to encompass the phrase “indirectly identifying” or “indirectlyidentified.”

ENDOGENOUS shall mean a material that a mammal naturally produces.ENDOGENOUS in reference to, for example and not limitation, the term“receptor,” shall mean that which is naturally produced by a mammal (forexample, and not limitation, a human) or a virus. By contrast, the termNON-ENDOGENOUS in this context shall mean that which is not naturallyproduced by a mammal (for example, and not limitation, a human) or avirus. For example, and not limitation, a receptor which is notconstitutively active in its endogenous form, but when manipulatedbecomes constitutively active, is most preferably referred to herein asa “non-endogenous, constitutively activated receptor.” Both terms can beutilized to describe both “in vivo” and “in vitro” systems. For example,and not limitation, in a screening approach, the endogenous ornon-endogenous receptor may be in reference to an in vitro screeningsystem. As a further example and not limitation, where the genome of amammal has been manipulated to include a non-endogenous constitutivelyactivated receptor, screening of a candidate compound by means of an invivo system is viable.

G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION PROTEIN, inthe context of the invention disclosed herein, each mean anon-endogenous protein comprising an endogenous, constitutively activateGPCR or a non-endogenous, constitutively activated GPCR fused to atleast one G protein, most preferably the alpha (α) subunit of such Gprotein (this being the subunit that binds GTP), with the G proteinpreferably being of the same type as the G protein that naturallycouples with endogenous GPCR. For example, and not limitation, in anendogenous state, if the G protein “Gsα” is the predominate G proteinthat couples with the GPCR, a GPCR Fusion Protein based upon thespecific GPCR would be a non-endogenous protein comprising the GPCRfused to Gsα; in some circumstances, as will be set forth below, anon-predominant G protein can be fused to the GPCR. The G protein can befused directly to the c-terminus of the constitutively active GPCR orthere may be spacers between the two.

HOST CELL shall mean a cell capable of having a Plasmid and/or Vectorincorporated therein. In the case of a prokaryotic Host Cell, a Plasmidis typically replicated as a autonomous molecule as the Host Cellreplicates (generally, the Plasmid is thereafter isolated forintroduction into a eukaryotic Host Cell); in the case of a eukaryoticHost Cell, a Plasmid is integrated into the cellular DNA of the HostCell such that when the eukaryotic Host Cell replicates, the Plasmidreplicates. Preferably, for the purposes of the invention disclosedherein, the Host Cell is eukaryotic, more preferably, mammalian, andmost preferably selected from the group consisting of Hek-293, Hek-293Tand COS-7 cells.

INDIRECTLY IDENTIFYING or INDIRECTLY IDENTIFIED means the traditionalapproach to the drug discovery process involving identification of anendogenous ligand specific for an endogenous receptor, screening ofcandidate compounds against the receptor for determination of thosewhich interfere and/or compete with the ligand-receptor interaction, andassessing the efficacy of the compound for affecting at least one secondmessenger pathway associated with the activated receptor.

INHIBIT or INHIBITING, in relationship to the term “response” shall meanthat a response is decreased or prevented in the presence of a compoundas opposed to in the absence of the compound.

INVERSE AGONISTS shall mean materials (e.g., ligand, candidatecompounds) which bind to either the endogenous form of the receptor orto the constitutively activated form of the receptor, and which inhibitthe baseline intracellular response initiated by the active form of thereceptor below the normal base level of activity which is observed inthe absence of agonists or partial agonists, or decrease GTP binding tomembranes. Preferably, the baseline intracellular response is inhibitedin the presence of the inverse agonist by at least 30%, more preferablyby at least 50%, and most preferably by at least 75%, as compared withthe baseline response in the absence of the inverse agonist.

KNOWN RECEPTOR shall mean an endogenous receptor for which theendogenous ligand specific for that receptor has been identified.

LIGAND shall mean an endogenous, naturally occurring molecule specificfor an endogenous, naturally occurring receptor.

MUTANT or MUTATION in reference to an endogenous receptor's nucleic acidand/or amino acid sequence shall mean a specified change or changes tosuch endogenous sequences such that a mutated form of an endogenous,non-constitutively activated receptor evidences constitutive activationof the receptor. In terms of equivalents to specific sequences, asubsequent mutated form of a human receptor is considered to beequivalent to a first mutation of the human receptor if (a) the level ofconstitutive activation of the subsequent mutated form of a humanreceptor is substantially the same as that evidenced by the firstmutation of the receptor; and (b) the percent sequence (amino acidand/or nucleic acid) homology between the subsequent mutated form of thereceptor and the first mutation of the receptor is at least about 80%,more preferably at least about 90% and most preferably at least 95%.Ideally, and owing to the fact that the most preferred cassettesdisclosed herein for achieving constitutive activation includes a singleamino acid and/or codon change between the endogenous and thenon-endogenous forms of the GPCR, the percent sequence homology shouldbe at least 98%.

NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurringmolecule specific for an endogenous naturally occurring ligand whereinthe binding of a ligand to a receptor activates an intracellularsignaling pathway.

ORPHAN RECEPTOR shall mean an endogenous receptor for which theendogenous ligand specific for that receptor has not been identified oris not known.

PHARMACEUTICAL COMPOSITION shall mean a composition comprising at leastone active ingredient, whereby the composition is amenable toinvestigation for a specified, efficacious outcome in a mammal (forexample, and not limitation, a human). Those of ordinary skill in theart will understand and appreciate the techniques appropriate fordetermining whether an active ingredient has a desired efficaciousoutcome based upon the needs of the artisan.

PLASMID shall mean the combination of a Vector and cDNA. Generally, aPlasmid is introduced into a Host Cell for the purposes of replicationand/or expression of the cDNA as a protein.

STIMULATE or STIMULATING, in relationship to the term “response” shallmean that a response is increased in the presence of a compound asopposed to in the absence of the compound.

VECTOR in reference to cDNA shall mean a circular DNA capable ofincorporating at least one cDNA and capable of incorporation into a HostCell.

The order of the following sections is set forth for presentationalefficiency and is not intended, nor should be construed, as a limitationon the disclosure or the claims to follow.

A. Introduction

Constitutively active forms of known G protein-coupled receptors,disclosed in the present patent document, can be obtained bysite-directed mutational methods, well-known to those skilled in theart. A constitutively active receptor useful for direct identificationof candidate compounds is preferably achieved by mutating the receptorat a specific location within an intracellular loop, most preferablywithin the intracellular loop three (IC3) region. Such mutation canproduce a non-endogenous receptor that is intended to be constitutivelyactivated, as evidenced by an increase in the functional activity of thereceptor, for example, an increase in the level of second messengeractivity. While standard methods of site-directed mutagenesis may beemployed, a preferred method is one that is disclosed in a co-pending,commonly assigned patent document U.S. application Ser. No. 09/170,496,filed Oct. 13, 1998 which is incorporated herein by reference.

Table B below lists known endogenous GPCRs that have been converted tonon-endogenous versions thereof, their respective G protein andendogenous ligand.

TABLE B Known GPCRs G Protein Endogenous Ligand 5HT-1A Gi Serotonin5HT-1B Gi Serotonin 5HT-1D Gi Serotonin 5HT-1E Gi Serotonin 5HT-1F GiSerotonin 5HT-2B Gi Serotonin 5HT-4A N/I Serotonin 5HT-4B N/I Serotonin5HT-4C N/I Serotonin 5HT-4D N/I Serotonin 5HT-4E N/I Serotonin 5HT-5AUnknown Serotonin 5HT-6 Gs Serotonin 5HT-7 Gs Serotonin AVPR1A GqArginine Vasopressin AVPRIB Gq Arginine Vasopressin AVPR2 Gs ArginineVasopressin BBR3 Gq Bombesin BDKR1 Gq Bradykinin BDKR2 Gq Bradykinin C3aN/I Anaphylatoxin C5a N/I Anaphylatoxin CB1 Gi Cannabinoid CB2 GiCannabinoid CCR2b Gi Monocyte chemoattractant (MCP) CCR3 Gi Eotaxin,Leukotactin-1, RANTES, MCP CCR5 Gi MIP-1α, MIP-1β, RANTES CCR8 Gi I-309,TARC, MIP-1β CCR9 N/I Thymus-expressed chemokine (TECK) CRFR1 GsCorticotropin-releasing- factor CXCR4 Gi SDF1 Dopamine D1 Gs DopamineDopamine D2 Gi Dopamine Dopamine D3 Gi Dopamine Dopamine D5 Gs DopamineETA Gq Endothelin ETB Gq Endothelin FPR1 N/I Formylpeptide FPRL1 N/Iformylpeptide GALR1 Gi Galanin GALR2 Gi/Gq Galanin GIP N/I Gastricinhibitory polypeptide mGluR1 Gq Glutamate GPR5 N/I Single C motif-1(SMC-1) GPR24 Gi/Gq Melanin Concentrating (also known as Hormone MCH orSLC-1) GRPR Gq Gastrin releasing peptide M1 Gq Acetylcholine M2 GiAcetylcholine M3 Gq Acetylcholine M4 Gi Acetylcholine M5 GqAcetylcholine MC3 Gs Melanocortin NK1R Gq Substance P NK2R GqNeurokinin-A NK3R Gq Neurokinin-B NMBR Gq Neuromedin B NPY5 GiNeuropeptide Y NTSR1 Gq Neurotensin NTSR2 Gq Neurotensin OPRD Gi OpiodOPRL1 Gi Opiod OPRK Gi Opiod OPRM Gi Opiod OPRM1A Gi Opiod OX₁R N/IOrexin OX₂R N/I Orexin PACAP Gs Pituitary adenylyl cyclase activatingpeptide PAF N/I Platelet activating factor PGE EP1 N/I Prostaglandin PGEEP2 Gs Prostaglandin PGE EP4 N/I Prostaglandin PTHR1 N/I Parathyroidhormone PTHR2 N/I Parathyroid hormone SCTR Gs Secretin SST1 GiSomatostatin SST2 Gi Somatostatin SST3 Gi Somatostatin SST4 GiSomatostatin SST5 Gi Somatostatin TSHR Gs Thyroid Stimulating HormoneVIPR Gs Vasoactive Intestinal Peptide VIPR2 Gs Vasoactive IntestinalPeptide Note: N/I means not indicated

B. Receptor Screening

Screening candidate compounds against a non-endogenous, constitutivelyactivated version of the known GPCRs disclosed herein allows for thedirect identification of candidate compounds which act at the cellsurface of the receptor, without requiring use of the receptor'sendogenous ligand. By determining areas within the body where theendogenous version of known GPCRs disclosed herein is expressed and/orover-expressed, it is possible to determine related disease/disorderstates which are associated with the expression and/or over-expressionof the receptor; such an approach is disclosed in this patent document.

Table C below lists the known GPCRs and tissues within the body areexpressed and/or over-expressed. The listed references provide supportfor such tissue expression.

TABLE C Known GPCRs Location of Expression Reference 5HT-1A N/I N/I5HT-1B Striatum Jin, H. et al., 267(9) J Biol Chem 5735 (1992) 5HT-1DCerebral cortex Weinshank, R. L. et al., 89(8) Proc Natl Acad Sci USA3630 (1992) 5HT-1E N/I N/I 5HT-1F Brain Adham, N. et al., 90 Proc NatlAcad Sci USA 408 (1993) 5HT-2B Various tissues, including Kursar, J. D.,46(2_Mol Brain Pharmacol 227 (1994) 5HT-4A Brain, Intestine and AtriumBlondel, O. et al., 70 J Nerochem 2252 (1998) 5HT-4B Brain, Intestineand Atrium Blondel, O. et al., 70 J Nerochem 2252 (1998) 5HT-4C Brain,Intestine and Atrium Blondel, O. et al., 70 J Nerochem 2252 (1998)5HT-4D Intestine Blondel, O. et al., 70 J Neurochem 2252 (1998) 5HT-4EBrain Claeysen, S. et al., 55(5) Mol Pharmacol 910 (1999) 5HT-5A BrainRees, S. et al., 355(3) FEBS Lett 242 (1994) 5HT-6 Caudate NucleusKohen, R. et al., 66(1) J Neurochem 47 (1996) 5HT-7 Brain, Coronaryartery Bard, J. A. et al., 268(31) J Biol Chem 23422 (1993) AVPR1A N/IN/I AVPR1B Pituitary Sugimoto, T. et al., 269(43) J. Biol. Chem 27088(1994) AVPR2 Lung, Kidney Fay, M. J., et al., 17(3) Peptides 477 (1996)BBR3 Testis, Lung carcinoma Fathi, Z. et al., 268(8) J. Biol. Chem. 5979(1993) BDKR1 N/I N/I BDKR2 N/I N/I C3a Lung, Spleen, Ovary, Ames, R. etal., 271(34) J. Placenta, Small Intestine and Biol. Chem 20231(1996)Brain C5a N/I N/I CB1 Brain Gerard, C. M. et al., 279 Biochem J. 129(1991) CB2 Spleen, Macrophage Munro, S. et al., 365(6441) Nature 61(1993) CCR2b N/I N/I CCR3 Endometrium Zhang, J. et al., 62(2) BiolReprod 404 (2000) CCR5 Thymus, Spleen Raport, C. J. et al., 271(29) JBiol Chem 17161 (1996) CCR8 Thymus, Spleen and Lymph Napolitano M. etal., Forum nodes (Geneva) 1999 Oct- Dec;9(4):315-24 CCR9 ThymusZaballos, A. et al., 162(10) J. Immunol 5671 (1999) CRFR1 Brain,Pituitary Perrin, M. H. et al., 133(6) Endocrinology 3058 (1993) CXCR4Colonic epithelial cells Jordan, N. J. et al., 104(8) J Clin Invest 1061(1999) Dopamine Caudate, Nucleus accumbens Dearry, A. et al., 347 NatureD1 and Olfactory tubercle 72 (1990) Dopamine Retina Dearry, A. et al.,11(5) Cell D2 Mol. Neurobiol. 437 (1991) Dopamine Brain Schmauss, C. etal., 90(19) D3 Proc Natl Acad Sci USA 8942 (1993) Dopamine BrainSunahara, R. K. et al., D5 350(6319) Nature 614 (1991) ETA Placenta,Uterus, Testis, Adachi, M. et al., 180(3) Adrenal gland Biochem BiophysRes Commun 1265 (1991) ETB N/I N/I FPR1 N/I N/I FPRL1 N/I N/I GALR1Hypothalamic Gundlach, A. L. et al., 863 paraventricular, Supraoptic AnnNY Acad Sci 241 nuclei (1998) GALR2 Hypothalamus, Fathi, Z. et al., 51Brain Res Hippocampus, Anterior Mol Brain Res 49 (1997) pituitary GIPN/I N/I mGluR1 Brain Stephan, D. et al., 35(12) Neuropharmacology 1649(1996) GPR5 Leukocyte cells Shan, L. et al., 268(3) Biochem Biophys ResCommun 938 (2000) GPR24 Fore-brain, Hypothalamus Kolakowki L F Jr. etal., (also 398(2-3) FEBS Lett 253 known (1996) as MCH or SLC-1) GRPRLung carcinoma cells Corjay, M. H. et al., 266 Jo Biol Chem 18771 (1991)M1 Heart, Pancreas and Neuronal Peralta, E. G. et al., Embo J. celllines 6(13) 3923 (1987) M2 Heart, Pancreas and Neuronal Peralta, E. G.et al., Embo J. cell lines 6(13) 3923 (1987) M3 Heart, Pancreas andNeuronal Peralta, E. G. et al., Embo J. cell lines 6(13) 3923 (1987) M4Heart, Pancreas and Neuronal Peralta, E. G. et al., Embo J. cell lines6(13) 3923 (1987) M5 Brain Bonner, T. I. et al., Neuron 1(5), 403 (1988)MC3 Brain, Placenta, Gut Gantz I. et al., 268(11) Jo Biol Chem 8246(1993) NK1R Spinal cord, Lung Taked, Y. et aL, 179(3) Biochem BiophysRes Commun 1232(1991) NK2R N/I N/I NK3R N/I N/I NMBR Lung carcinomacells Corjay, M. H. et al., 266 Jo Biol Chem 18771 (1991) NPY5Hypothalamus Gerald, C. et al., 382 Nature 168 (1996) NTSR1 Brain, Smallintestine Vita, N. et al., 17 (1-2) FEBS Lett 139 (1993) NTSR2 N/I N/IOPRD Peripheral blood Wick, M. J. et al., 64(1) J. lymphocytesNeuroimmunol 29 (1996) OPRL1 N/I N/I OPRK Placenta, Brain Manson, E. etal., 202(3) Biochem Biophys Res Commun 1431 (1994) OPRM N/I N/I OPRM1AN/I N/I OX₁R Hypothalamus Sakurai T. et al., 92 Cell 573 (1998) OX₂RHypothalamus Sakurai T. et al., 92 Cell 573 (1998) PACAP Brain Ogi, K.et al., 196(3) Biochem Biophys Res Commun 1511 (1993) PAF N/I N/I PGEEP1 Kidney Watabe, A. et al., 268(27) J Biol Chem 20175 (1993) PGE EP2Small Intestine Bastien, L. et al., 269(16) J. Biol. Chem 11873 (1994)PGE EP4 N/I N/I PTHR1 Bone, Kidney Schipani, E. et al., 132(5)Endocrinology 2157 (1993) PTHR2 Brain, Pancreas Usdin, T. B. et al.,270(26) J. Biol Chem 15455 (1995) SCTR Pancreas, Intestine Chow, B. K.,212(1) Biochem Biophys Res Commun 204 (1995) SST1 Jejunum, StomachYamada Y. et al., 89 Proc Natl Acad Sci USA 251 (1992) SST2 Cerebrum,Kidney Yamada Y. et al., 89 Proc Natl Acad Sci USA 251 (1992) SST3Brain, Pancreatic islet Yamada Y. et al., 6 Mol Endocrinol 2136 (1992)SST4 Fetal, Adult Brain, Lung Rohser L. et al., 90(9) Pro Natl Acad SciUSA 4146 (1993) SST5 Pituitary Panetta R. et al., 45(3) mol Pharmacol417 (1994) TSHR Retro-orbital tissues, Feliciello A. et al 342 LancetExophthalmos 337 (1993) VIPR Lung Sreedharan, S. P. et al., 193(2)Biochem Biophys Res Commun 546 (1993) VIPR2 Skeletal muscle Adamou, J.E. et al., 209(2) Biochem Biophys Res Commun 385 (1995) Note: N/I meansnot indicated

Creation of a non-endogenous version of a known GPCR that may evidenceconstitutive activity is most preferably based upon the distance from aproline residue located within TM6 of the GPCR; this technique isdisclosed in co-pending and commonly assigned patent document U.S. Ser.No. 09/170,496, incorporated herein by reference. This technique is notpredicated upon traditional sequence “alignment” but rather a specifieddistance from the aforementioned TM6 proline residue. By mutating theamino acid residue located 16 amino acid residues from this residue(presumably located in the IC3 region of the receptor) to, mostpreferably, a lysine residue, such activation may be obtained. Otheramino acid residues may be used for the mutation, but lysine is mostpreferred.

D. Disease/Disorder Identification and/or Selection

As will be set forth in greater detail below, most preferably inverseagonists, partial agonists and agonists in the form of small moleculechemical compounds to the non-endogenous, constitutively activated GPCRcan be identified by the methodologies of this invention. Such compoundsare ideal candidates as lead modulators in drug discovery programs fortreating diseases or disorders associated with a particular receptor.The ability to directly identify such compounds to the GPCR, in theabsence of use of the receptor's endogenous ligand, allows for thedevelopment of pharmaceutical compositions.

Preferably, in situations where it is unclear what disease or disordermay be associated with a receptor; the DNA sequence of the known GPCR isused to make a probe for (a) dot-blot analysis against tissue-mRNA,and/or (b) RT-PCR identification of the expression of the receptor intissue samples. The presence of a receptor in a tissue source, or adiseased tissue, or the presence of the receptor at elevatedconcentrations in diseased tissue compared to a normal tissue, can bepreferably utilized to identify a correlation with a treatment regimen,including but not limited to, a disease associated with that disease.Receptors can equally well be localized to regions of organs by thistechnique. Based on the known functions of the specific tissues to whichthe receptor is localized, the putative functional role of the receptorcan be deduced.

E. Screening of Candidate Compounds

1. Generic GPCR Screening Assay Techniques

When a G protein receptor becomes constitutively active, it binds to a Gprotein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the binding of GTP tothe G protein. The G protein then acts as a GTPase and slowly hydrolyzesthe GTP to GDP, whereby the receptor, under normal conditions, becomesdeactivated. However, constitutively activated receptors continue toexchange GDP to GTP. A non-hydrolyzable analog of GTP, [³⁵S]GTPγS, canbe used to monitor enhanced binding to membranes which expressconstitutively activated receptors. It is reported that [³⁵S]GTPγS canbe used to monitor G protein coupling to membranes in the absence andpresence of ligand. An example of this monitoring, among other exampleswell-known and available to those in the art, was reported by Traynorand Nahorski in 1995. The preferred use of this assay system is forinitial screening of candidate compounds because the system isgenerically applicable to all G protein-coupled receptors regardless ofthe particular G protein that interacts with the intracellular domain ofthe receptor.

2. Specific GPCR Screening Assay Techniques

Once candidate compounds are identified using the “generic” Gprotein-coupled receptor assay (i.e., an assay to select compounds thatare agonists, partial agonists, or inverse agonists), further screeningto confirm that the compounds have interacted at the receptor site ispreferred. For example, a compound identified by the “generic” assay maynot bind to the receptor, but may instead merely “uncouple” the Gprotein from the intracellular domain.

a. Gs, Gz and Gi.

Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), on theother hand, inhibit this enzyme. Adenylyl cyclase catalyzes theconversion of ATP to cAMP; thus, constitutively activated GPCRs thatcouple the Gs protein are associated with increased cellular levels ofcAMP. On the other hand, constitutively activated GPCRs that couple Gi(or Gz, Go) protein are associated with decreased cellular levels ofcAMP. See, generally, “Indirect Mechanisms of Synaptic Transmission,”Chpt. 8, From Neuron To Brain (3^(rd) Ed.) Nichols, J. G. et al eds.Sinauer Associates, Inc. (1992). Thus, assays that detect cAMP can beutilized to determine if a candidate compound is, e.g., an inverseagonist to the receptor (i.e., such a compound would decrease the levelsof cAMP). A variety of approaches known in the art for measuring cAMPcan be utilized; a most preferred approach relies upon the use ofanti-cAMP antibodies in an ELISA-based format. Another type of assaythat can be utilized is a second messenger reporter system assay.Promoters on genes drive the expression of the proteins that aparticular gene encodes. Cyclic AMP drives gene expression by promotingthe binding of a cAMP-responsive DNA binding protein or transcriptionfactor (CREB) that then binds to the promoter at specific sites calledcAMP response elements and drives the expression of the gene. Reportersystems can be constructed which have a promoter containing multiplecAMP response elements before the reporter gene, e.g., β-galactosidaseor luciferase. Thus, a constitutively activated Gs-linked receptorcauses the accumulation of cAMP that then activates the gene andexpression of the reporter protein. The reporter protein such asβ-galactosidase or luciferase can then be detected using standardbiochemical assays (Chen et al. 1995).

b. Go and Gq.

Gq and Go are associated with activation of the enzyme phospholipase C,which in turn hydrolyzes the phospholipid PIP₂, releasing twointracellular messengers: diacycloglycerol (DAG) and inistol1,4,5-triphoisphate (IP₃). Increased accumulation of PP₃ is associatedwith activation of Gq- and Go-associated receptors. See, generally,“Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron ToBrain (3^(rd) Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc.(1992). Assays that detect IP₃ accumulation can be utilized to determineif an candidate compound is, e.g., an inverse agonist to a Gq- orGo-associated receptor (i.e., such a compound would decrease the levelsof IP₃). Gq-associated receptors can also be examined using an AP1reporter assay in that Gq-dependent phospholipase C causes activation ofgenes containing AP1 elements; thus, activated Gq-associated receptorswill evidence an increase in the expression of such genes, wherebyinverse agonists thereto will evidence a decrease in such expression,and agonists will evidence an increase in such expression. Commerciallyavailable assays for such detection are available.

3. Ligand-Based Confirmation Assays

The candidate compounds directly identified using the techniques (orequivalent techniques) above are then, most preferably, verified using aligand-based verification assay, such as the one set forth in theprotocol of Example 8. The importance here is that the candidatecompound be directly identified; subsequent confirmation, if any, usingthe endogenous ligand, is merely to confirm that the directly identifiedcandidate compound has targeted the receptor.

For example, sumatriptan is a well-known agonist of the 5-HT1B and5-HT1D receptors, while naltrindole is a well-known antagonist to theOPMID receptor. Accordingly, an agonist (sumatriptan) and/or antagonist(naltrindole) competitive binding assay(s) can be used to confirm thatthose candidate compounds directly identified using a ligand independentscreening technique comprising non-endogenous, constitutively activated5-HT1B or 5-HT1D, and non-endogenous constitutively activated OPM1D,respectfully, may be used for confirmatory purposes. Those skilled inthe art are credited with the ability to select techniques forligand-based confirmation assays.

4. GPCR Fusion Protein

The use of a non-endogenous, constitutively activated GPCR, for use inscreening of candidate compounds for the direct identification ofinverse agonists, agonists and partial agonists, provides an interestingscreening challenge in that, by definition, the receptor is active evenin the absence of an endogenous ligand bound thereto. Thus, in order todifferentiate between, e.g., the non-endogenous receptor in the presenceof a candidate compound and the non-endogenous receptor in the absenceof that compound, with an aim of such a differentiation to allow for anunderstanding as to whether such compound may be an inverse agonist,agonist, partial agonist or has no affect on such a receptor, it ispreferred that an approach be utilized that can enhance suchdifferentiation. A preferred approach is the use of a GPCR FusionProtein.

Generally, once it is determined that a non-endogenous GPCR has beenconstitutively activated using the assay techniques set forth above (aswell as others), it is possible to determine the predominant G proteinthat couples with the endogenous GPCR. Coupling of the G protein to theGPCR provides a signaling pathway that can be assessed. Because it ismost preferred that screening take place by use of a mammalianexpression system, such a system will be expected to have endogenous Gprotein therein. Thus, by definition, in such a system, thenon-endogenous, constitutively activated GPCR will continuously signal.In this regard, it is preferred that this signal be enhanced such thatin the presence of, e.g., an inverse agonist to the receptor, it is morelikely that it will be able to more readily differentiate, particularlyin the context of screening, between the receptor when it is contactedwith the inverse agonist.

The GPCR Fusion Protein is intended to enhance the efficacy of G proteincoupling with the non-endogenous GPCR. The GPCR Fusion Protein ispreferred for screening with a non-endogenous, constitutively activatedGPCR because such an approach increases the signal that is mostpreferably utilized in such screening techniques. This is important infacilitating a significant “signal to noise” ratio; such a significantratio is preferred for the screening of candidate compounds as disclosedherein.

The construction of a construct useful for expression of a GPCR FusionProtein is within the purview of those having ordinary skill in the art.Commercially available expression vectors and systems offer a variety ofapproaches that can fit the particular needs of an investigator. Thecriteria of importance for such a GPCR Fusion Protein construct is thatthe endogenous GPCR sequence and the G protein sequence both be in-frame(preferably, the sequence for the endogenous GPCR is upstream of the Gprotein sequence) and that the “stop” codon of the GPCR must be deletedor replaced such that upon expression of the GPCR, the G protein canalso be expressed. The GPCR can be linked directly to the G protein, orthere can be spacer residues between the two (preferably, no more thanabout 12, although this number can be readily ascertained by one ofordinary skill in the art). Use of a spacer is preferred (based uponconvenience) in that some restriction sites that are not used will,effectively, upon expression, become a spacer. Most preferably, the Gprotein that couples to the non-endogenous GPCR will have beenidentified prior to the creation of the GPCR Fusion Protein construct.Because there are only a few G proteins that have been identified, it ispreferred that a construct comprising the sequence of the G protein(i.e., a universal G protein construct) be available for insertion of anendogenous GPCR sequence therein; this provides for efficiency in thecontext of large-scale screening of a variety of different endogenousGPCRs having different sequences.

F. Co-Transfection of a Target Gi Coupled GPCR with a Signal-Enhancer GsCoupled GPCR (cAMP Based Assays)

A Gi coupled receptor is known to inhibit adenylyl cyclase, and,therefore, decrease the level of cAMP production, which can makeassessment of cAMP levels challenging. An effective technique inmeasuring the decrease in production of cAMP as an indication ofconstitutive activation of a receptor that predominantly couples Gi uponactivation can be accomplished by co-transfecting a signal enhancer,e.g., a non-endogenous, constitutively activated receptor thatpredominantly couples with Gs upon activation (e.g., TSHR-A623I,disclosed below), with the Gi linked GPCR (such a technique isexemplified herein with the Gi coupled receptor, GPR24). As is apparent,constitutive activation of a Gs coupled receptor can be determined basedupon an increase in production of cAMP. Constitutive activation of a Gicoupled receptor leads to a decrease in production cAMP. Thus, theco-transfection approach is intended to advantageously exploit these“opposite” affects. For example, co-transfection of a non-endogenous,constitutively activated Gs coupled receptor (the “signal enhancer”)with the endogenous Gi coupled receptor (the “target receptor”) providesa baseline cAMP signal (i.e., although the Gi coupled receptor willdecrease cAMP levels, this “decrease” will be relative to thesubstantial increase in cAMP levels established by constitutivelyactivated Gs coupled signal enhancer). By then co-transfecting thesignal enhancer with a constitutively activated version of the targetreceptor, cAMP would be expected to further decrease (relative to baseline) due to the increased functional activity of the Gi target (i.e.,which decreases cAMP).

Screening of candidate compounds using a cAMP based assay can then beaccomplished, with two provisos: first, relative to the Gi coupledtarget receptor, “opposite” effects will result, i.e., an inverseagonist of the Gi coupled target receptor will increase the measuredcAMP signal, while an agonist of the Gi coupled target receptor willdecrease this signal; second, as would be apparent, candidate compoundsthat are directly identified using this approach should be assessedindependently to ensure that these do not target the signal enhancingreceptor (this can be done prior to or after screening against theco-transfected receptors).

G. Medicinal Chemistry

Generally, but not always, direct identification of candidate compoundsis preferably conducted in conjunction with compounds generated viacombinatorial chemistry techniques, whereby thousands of compounds arerandomly prepared for such analysis. Generally, the results of suchscreening will be compounds having unique core structures; thereafter,these compounds are preferably subjected to additional chemicalmodification around a preferred core structure(s) to further enhance themedicinal properties thereof. Such techniques are known to those in theart and will not be addressed in detail in this patent document.

H. Pharmaceutical Compositions

Candidate compounds selected for further development can be formulatedinto pharmaceutical compositions using techniques well known to those inthe art. Suitable pharmaceutically-acceptable carriers are available tothose in the art; for example, see Remington's Pharmaceutical Sciences,16^(th) Edition, 1980, Mack Publishing Co., (Oslo et al., eds.).

I. Other Utility

Although a preferred use of the non-endogenous version of the knownGPCRs disclosed herein may be for the direct identification of candidatecompounds as inverse agonists, agonists or partial agonists (preferablyfor use as pharmaceutical agents), these versions of known GPCRs canalso be utilized in research settings. For example, in vitro and in vivosystems incorporating GPCRs can be utilized to further elucidate andbetter understand the roles these receptors play in the human condition,both normal and diseased, as well as understanding the role ofconstitutive activation as it applies to understanding the signalingcascade. The value in non-endogenous known GPCRs is that their utilityas a research tool is enhanced in that, because of their uniquefeatures, non-endogenous known GPCRs can be used to understand the roleof these receptors in the human body before the endogenous ligandtherefor is identified. Other uses of the disclosed receptors willbecome apparent to those in the art based upon, inter alia, a review ofthis patent document.

EXAMPLES

The following examples are presented for purposes of elucidation, andnot limitation, of the present invention. While specific nucleic acidand amino acid sequences are disclosed herein, those of ordinary skillin the art are credited with the ability to make minor modifications tothese sequences while achieving the same or substantially similarresults as reported below. The traditional approach to application orunderstanding of sequence cassettes from one sequence to another (e.g.from rat receptor to human receptor or from human receptor A to humanreceptor B) is generally predicated upon sequence alignment techniqueswhereby the sequences are aligned in an effort to determine areas ofcommonality. The mutational approaches disclosed herein do not rely upona sequence alignment approach but are instead based upon an algorithmicapproach and a positional distance from a conserved proline residuelocated within the TM6 region of GPCRs. Once this approach is secured,those in the art are credited with the ability to make minormodifications thereto to achieve substantially the same results (i.e.,constitutive activation) disclosed herein. Such modified approaches areconsidered within the purview of this disclosure

Example 1

Preparation of Endogenous Known GPCRS

A. Expression By Standard PCR

PCR was performed using a specific cDNA as template and rTth polymerase(Perkin Elmer) with the buffer system provided by the manufacturer, 0.25μM of each primer, and 0.2 mM of each 4 nucleotides.

The resulting PCR fragment was digested with the respective restrictionsites and cloned into a pCMV expression vector. Nucleic acid and aminoacid sequences were thereafter determined and verified. See Table Dbelow:

TABLE D 5′ Primer 3′ Primer Receptor Cycle Conditions (SEQ.ID.NO.) and(SEQ.ID.NO.) and Identifier Template Min (′), Sec (″) Restriction siteRestriction site 5HT-1A Genomic DNA 94° for 1′ CGGAAGCTTAGC CCGGAATTCCTG60° C. for 1′ CATGGATGTGCT GCGGCAGAAGTT 72° for 1′30″ CAGCCCTGGTCAACACTTAATG (2); (1); HindIII EcoRI 5HT-1B Genomic DNA 94° for 1′TCCAAGCTTGGG GGCGAATTCACTT 60° C. for 1′ GCGAGGAGAGCC GTGCACTTAAAA72° for 1′30″ ATGGAGGA (3); CGTATCAGTT (4); HindIII EcoRI 5HT-1D GenomicDNA 94° for 1′ ATCTACCATGTC ATAGAATTCGGA 60° C. for 1′ CCCACTGAACCAGGCCTTCCGGAA 72° for 1′30″ GTCAGC (5) AGGGACAA (6); EcoRI 5HT-1E GenomicDNA 94° for 1′ CCACAGTGTCGA CAGTATGCTCTCG 60° C. for 1′ CTGAAACAAGGGGCATCTAATGAG 72° for 2′10″ AAACATGAAC (8) (7); SalI 5HT-1E Genomic DNA94° for 1′ ATCACCATGGAT TTAGGATCCACAT 60° C. for 1′ TTCTTAAATTCATCGACATCGCACA 72° for 2′10″ CTGATC (9) AGCTTTTG (10); BamHI 5HT-2B UteruscDNA 94° for 1′ GAAAAGCTTGCC GTTGGATCCTACA 60° C. for 1′ ATGGCTCTCTCTTTAACTAACTTGCT 72° for 1′30″ ACAGAGTGTCTG CTTCAGTTT (12); (11); HindIIIBamHI 5HT-4A Brain cDNA 94° for 1′ ATCACCATGGAC CCTGAATTCGAA 60° C. for1′ AAACTTGATGCT GCATGATTCCAG 72° for 1′30″ AATGTGAG (13) GGATTCTGG (14);EcoRI 5HT-4B Brain cDNA 94° for 1′ ATCACCATGGAC AGGGAATTCAGT 60° C. for1′ AAACTTGATGCT GTCACTGGGCTG 72° for 1′30″ AATGTGAG (15) AGCAGCCAC (16);EcoRI 5HT-4C Brain cDNA 94° for 1′ ATCACCATGGAC TTGGAATTCGGAT 60° C. for1′ AAACTTGATGCT GGTTTGGTCAATC 72° for 1′30″ AATGTGAG (17) TTCTCTTC (18);EcoRI 5HT-4D 5HT-4E DNA 94° for 1′ ATCACCATGGAC AGGGAATTCAAA 60° C. for1′ AAACTTGATGCT TCTTAGTACATGT 72° for 2″ AATGTGAG (19) GTGGATCCATTA AT(20); EcoRI 5HT-4E Brain cDNA 94° for 1′ ATCACCATGGAC TCAGAATTCGAC60° C. for 1′ AAACTTGATGCT AGGAACTGGTCT 72° for 1′15″ AATGTGAG (21)ATTGCAGAA (22); EcoRI 5HT-5A Brain cDNA 94° for 1′ CCTAAGCTTGCCTCTGAATTCGTGT 60° C. for 1′ ATGGATTTACCA TGCCTAGAAAAG 72° for 2′10″GTGAACCTAACC AAGTTCTTGA TCC (23); HindIII (24); EcoRI 5HT-6 Seealternative See alternative See alternative See alternative approachbelow approach below approach below approach below 5HT-7 Brain cDNA94° C. for 1′ AGCGGAATTCGG TTTCGGATCCATT 72° C. for 2″ CGGCGCGATGATGTTCTGCTTTCAA GGACGTT (25); TCAT (26); BamHI EcoRI AVPR1A Seealternative See alternative See alternative See alternative approachbelow approach below approach below approach below AVPR1A Seealternative See alternative See alternative See alternative variantapproach below approach below approach below approach below AVPR1B Seealternative See alternative See alternative See alternative approachbelow approach below approach below approach below AVPR2 IMAGE 301449pfu PCR CAGGAATTCAGA AGCGGATCCCGA 94° for 1′ ACACCTGCCCCA TGAAGTGTCCTTG63° C. for 1′ GCCCCAC (27); GCCAGGGA (28); 72° for 2″ EcoRI BamHI BBR3Uterus cDNA 94° C. for 1′ ACAGAATTCAGA CATGGATCCTTGA 56° C. for 1′AGAAATGGCTCA AAAGCTAGAAAC 72° C. for 1′20″ AAGGCA (29); TGTCC (30);BamHI EcoRI BDKR1 IMAGE 1472696 pfu PCR TGTAAGCTTCAG GCTGGATCCATTC94° for 1′ GTCACTGTGCAT CGCCAGAAAAGT 65° C. for 1′ GGCATCATC (31);TGGAAGATTTC 72° for 2″ HindIII (32); BamHI BDKR2 IMAGE 1682455 pfu PCRACTAAGCTTCCA GTTGAATTCCTGT 94° for 1′ AATGTTCTCTCCC CTGCTCCCTGCCC 65° C.for 1′ TGGAAGATA (33) AGTCCTG (34); 72° for 2″ HindIII EcoRI C3a GenomicDNA 94° for 1′ CAGAAGCTTAGC ACAGGATCCCAC 65° C. for 1′ AATGGCGTCTTTAGTTGTACTATTT 72° for 1′30″ CTCTGCTG (35); CTTTCTGAAATG HindIII (36);BamHI C5a Thymus 94° for 1′ GGGAAGCTTAGG TGTGAATTCCACT 65° C. for 1′AGACCAGAACAT GCCTGGGTCTTCT 72° for 1′10″ GAACTCCTTC GGGCCAT (38); (37);HindIII EcoRI CB1 EST 01536 pfu PCR GGGAAGCTTTCT TCAGAATTCCAG 94° for 1′CAGTCATTTTGA AGCCTCGGCAGA 65° C. for 1′ GCTCAGCC (39); CGTGTCTGT (40);72° for 2′30″ HindIII EcoRI CB2 IMAGE 1301708 pfu PCR CAAAAGCTTCTAGCCGAATTCGCA 94° for 1′ GACAAGCTCAGT ATCAGAGAGGTC 60° C. for 1′GGAATCTGA (41); TAGATCTCTG 72° for 2″ HindIII (42); EcoRI CCR2b GenomicDNA 94° for 1′ GACAAGCTTCCC CTCGGATCCTAA 60° C. for 1′ CAGTACATCCACACCAGCCGAGAC 72° for 1′10″ AACATGC (43); TTCCTGCTC (44); HindIII BamHICCR3 Genomic DNA 94° for 1′ ATCGCCATGACA TCTGAATTCAAAC 60° C. for 1′ACCTCACTAGAT ACAATAGAGAGT 72° for 1′10″ ACAGTTGAG (45) TCCGGCTC (46);EcoRI CCR5 Genomic DNA 94° for 1′ GCAAAGCTTGGA TCCGGATCCCAA 62° C. for1′ ACAAGATGGATT GCCCACAGATAT 72° for 1′10″ ATCAAGTGTC TTCCTGCTC (48);(47); HindIII BamHI CCR8 Genomic DNA 94° for 1′ TGAAAGCTTCCCTGAGAATTCCAA 60° C. for 1′ GCTGCCTTGATG AATGTAGTCTAC 72° for 1′10″GATTATAC (49); GCTGGAGGAA HindIII (50); EcoRI CCR9 Genomic DNA 94° for1′ ATCACCATGACA GACGAATTCGAG 60° C. for 1′ CCCACAGACTTC GGAGAGTGCTCC72° for 1′10″ ACAAGCCCTATT TGAGGTTGT (52); CCTAACATGGCT EcoRIGATGACTATGG (51) CRFR1 Pituitary cDNA 94° for 1′ ATCACCATGGGACGGGAATTCGAC 65° C. for 1′ GGGCACCCGCAG TGCTGTGGACTGC 72° for 1′20″CTCCGT (53) TTGATGCT (54); EcoRI CXCR4 Genomic DNA 94° for 1′ATCACCATGGAG TCTGAATTCGCTG 65° C. GGGATCAGTATA GAGTGAAAACTT 72° for 1′TACACTTCAGAT GAAGACTCAG AACTACACCGAG (56); EcoRI GAAATG (55) DopamineSee alternative See alternative See alternative See alternative D1approach below approach below approach below approach below Dopamine Seealternative See alternative See alternative See alternative D2 approachbelow approach below approach below approach below Dopamine Brain cDNA94° C. for 1′ AAGAAGCTTGGC GGCTCTAGAAAT D3 62° for 1′20″ ATCACGCACCTCGGGTACAAAGAG 72° C. for 1′20″ CTCTGG(57); TGTT (58); HindIII XbaIDopamine Genomic DNA See alternative See alternative See alternative D5approach below approach below approach below ETA See alternative Seealternative See alternative See alternative approach below approachbelow approach below approach below ETB IMAGE 1086987 pfu PCRCGGAAGCTTCTG CTTGGATCCAGAT 94° for 1′ GAGCAGGTAGCA GAGCTGTATTTAT 60° C.for 1′ GCATG (59); TACTGGAACG 72° for 2′20″ HindIII (60); BamHI FPR1IMAGE 2153284 94° C. for 1′ ATCACCATGGAG CCCGAATTCCTTT 63° for 1′ACAAATTCCTCT GCCTGTAACTCCA 72° C. for 2′30″ CTCCCC (61) CCTCTGC (62);EcoRI FPRL1 Genomic DNA 94° C. for 1′ GCAAAGCTTGCT CCAGAATTCCATT 65° for1′ GCTGGCAAGATG GCCTGTAACTCA 72° C. for 1′10″ GAAACCAAC (63); GTCTCTGC(64); HindIII EcoRI GALR1 Stomach cDNA 94° C. for 1′ CCGGAATTCGCCGCAGGATCCTTAT 60° for 1′20″ GGGACAGCCCCG CACACATGAGTA 72° C. for 1′20″CGGGCC (65); CAATTGGT (66); EcoRI BamHI GALR2 Hippocampus 94° C. for 1′GGCGAATTCGGG GTGGGATCCCAG cDNA 62° for 1′20″ GTCAGCGGCACC CGCGCCCGCTAA72° C. for 1′20″ ATGAACG (67); GTGCT (68); BamHI EcoRI GIP Brain cDNA94° for 1′ CAGAAGCTTCGC CGCGAATTCGCA 65° C. for 1′ CGCCCTCACGATGTAACTTTCCAAC 72° for 1′30″ GACTAC (69); TCCCGGCT (70); HindIII EcoRImGluR1 See alternative See alternative See alternative See alternativeapproach below approach below approach below approach below GPR5 GenomicDNA 94° C. for 1′ TATGAATTCAGA TCCGGATCCACCT 64° for 1′ TGCTCTAAACGTGCACCTGCGCCT 72° C. for 1′30″ CCCTGC (71); GCACC (72); BamHI EcoRI GPR24(also See alternative See alternative See alternative See alternativeknown as approach below approach below approach below approach below MCHor SLC-1) GRPR Stomach cDNA 94° C. for 1′ AGGAAGCTTTTA CCGGAATTCAAG 56°for 1′20″ GGTGGGAAAAA GGGCAAAATCAA 65° C. for 1′20″ AAATCTA (73);GGGTCAA (74); HindIII EcoRI M1 Genomic DNA 94° C. for 1′ GCCAAGCTTAGCGGAGAATTCGCA 60° for 1′ CACCATGAACAC TTGGCGGGAGGG 72° C. for 1′50″TTCAGCCC (75); AGTGCGGTG (76); HindIII EcoRI M2 Genomic DNA 94° C. for1′ ATCACCATGAAT GATGAATTCCCTT 60° for 1′ AACTCAACAAAC GTAGCGCCTATGT72° C. for 1′50″ TCCTCTAAC (77) TCTTATA (78); EcoRI M3 Genomic DNA94° C. for 1′ ATCACCATGACC CTCGAAATTCCA 60° for 1′ TTGCACAATAACAGGCCTGCTCGG 72° C. for 1′50″ AGTACAAC (79) GTGCGCGCT (80); EcoRI M4Genomic DNA 94° C. for 1′ ATCACCATGGCC GCCGAATTCCCTG 60° for 1′AACTTCACACCT GCAGTGCCGATG 72° C. for 1′50″ GTCAA (81) TTCCGATA (82);EcoRI M5 Genomic DNA 94° C. for 1′ ATCACCATGGAA GACGGATCCGGG 60° for 1′GGGGATTCTTAC TAGCTTGCTGTTC 72° C. for 1′50″ CACAAT (83) CCCTGCCA (84);BamHI MC3 Genomic DNA 94° C. for 1′ CAGGAATTCTGA AATGGATCCTATC 54° for1′30″ CAGCAATGAATG CCAAGTTCATGCC 72° C. for 1′20″ CTTCGT (85); GTTGCAG(86); EcoRI BamHI NK1R Brain cDNA 94° C. for 1′ AGTAAGCTTTACTGTGAATTCGGA 65° for 1′ GCCTAGCTTCGA GAGCACATTGGA 72° C. for 1′50″AATGGAT (87); GGAGAAGCT (88); HindIII EcoRI NK2R Uterus cDNA 94° C. for1′ TCCAAGCTTAGA AACGAATTCAAT 65° for 1′ AGCAGCCATGGG TTCAACATGAGTT72° C. for 1′50″ GACCTGTGACA TTGGTGGGGG (89); HindIII (90); EcoRI NK3RBrain cDNA 94° C. for 1′ ATCTGCAGACCG ATGGGATCCAGA 65° for 1′20″GTGGCGATGGCC ATATTCATCCACA 72° C. for 1′20″ ACT (91) GAGGTATAGG (92);BamHI NMBR Brain cDNA 94° C. for 1′ TGAGAATTCCAG GTTGGATCCAGG 65° for1′20″ CGGACTCTGCTG TAGTGAGTTGAA 72° C. for 1′20″ GAAAGGA (93); TGGCCA(94); EcoRI BamHI NPY5 Genomic DNA 94° C. for 1′ GGAAAGCTTCAAGGAGGATCCAGT 54° for 1′30″ GAAAGACTATAA GAGAATTATTAC 72° C. for 1′20″TATGGAT (95); ATATGAAG (96); HindIII BamHI NTSR1 See alternative Seealternative See alternative See alternative approach below approachbelow approach below approach below NTSR2 See alternative Seealternative See alternative See alternative approach below approachbelow approach below approach below OPRD Brain cDNA 94° C. for 1′CGGAAGCTTGCA GCCGAATTCGGC 65° for 1′ GCCATGGAACCG GGCAGCGCCACC 72° C.for 1′15 GCCCCCTCC (97); GCCGGGACC (98); HindIII EcoRI OPRL1 Brain cDNA94° C. for 1′ AGTAAGCTTGCA GCCGAATTCTGC 65° for 1′ GGGCAGTGGCATGGGCCGCGGTAC 72° C. for 1′15″ GGAGCCC (99); CGTCTCAGA (100); HindIIIEcoRI OPRK Brain cDNA 94° C. for 1′ TTTAAGCTTGCA CTACTGGTTTATT 65° for1′ GCACTCACCATG CATCCCATCGATG 72° C. for 1′15″ GAATCCCCGAT TC (102);(101); HindIII OPRM See alternative See alternative See alternative Seealternative approach below approach below approach below approach belowOPRM1A See alternative See alternative See alternative See alternativeapproach below approach below approach below approach below OX₁R Seealternative See alternative See alternative See alternative approachbelow approach below approach below approach below OX₂R Brain cDNA94° C. for 1′ ACCAAGCTTGAG CAGGGATCCTTGT 65° for 1′ CCCGTGATGTCCCATATGAATAAA 72° C. for 1′20″ GGCACC (103); TATT (104); BamHI HindIIIPACAP Fetal Brain cDNA 94° C. for 1′ AGTAAGCTTGGC CATGAATTCGGT 65° for1′ CAAGAAGTGTCA GGCCAGATTGTC 72° C. for 1′30″ TGGCTGGTG AGCAGGGAG (105);HindIII (106); EcoRI PAF Genomic DNA 94° C. for 1′ CTGAAGCTTCCACAGGAATTCATTT 63° for 1′ GCCCACAGCAAT TTGAGGGAATTG 72° C. for 2′30″GGAGCCA (107); CCAGGGATCTG HindIII (108); EcoRI PGE EP1 cDNA clone pfuPCR ATCGCCATGAGC TTGGAATTCGAA 94° C. for 1′ CCTTGCGGGCCC GTGGCTGAGGCC63° for 1′ CTCAA (109) GCTGTGCCG (110); 72° C. for 2′30″ EcoRI PGE EP2Thymus cDNA 94° C. for 1′ GCAAAGCTTTTC CTGGAATTCAAG 63° for 1′CAGGCACCCCAC GTCAGCCTGTTTA 72° C. for 2′30″ CATGGGC (111); CTGGCATC(112); HindIII EcoRI PGE EP4 cDNA clone pfu PCR ATCATCATGTCCTGCGAATTCTATA 94° C. for 1′ ACTCCCGGGGTC CATTTTTCTGATA 60° for 1′ AAT(113) AGTTCAGTGTT 72° C. for 2′30″ (114); EcoRI PTHR1 See alternativeSee alternative See alternative See alternative approach below approachbelow approach below approach below PTHR2 Brain cDNA 94° C. for 1′CTGAAGCTTCCT CGAGAACATCCT 65° for 1′ ACAGCCGTTCCG CAGTTTCTCCTTG 72° C.for 1′50″ GGCATG (115); G (116) HindIII SCTR Small Intestine 94° C. for1′ GGGAAGCTTGCG AGCGAATTCGAT 65° for 1′ GGCACCATGCGT GATGCTGGTCCTG72° C. for 1′45″ CCCCACCT (117); CAGGTGCC (118); HindIII EcoRI SST1Genomic DNA 94° C. for 1′ GCCGAATTCAGC CAGGGATCCTGC 65° for 1′20″TGGGATGTTCCC GTGGCCCGGGCT 72° C. for 1′20″ CAATGGC (119); CAGAGCG (120);EcoRI BamHI SST2 See alternative See alternative See alternative Seealternative approach below approach below approach below approach belowSST3 Genomic DNA 94° C. for 1′ ACGGAATTCCCC TGGGATCCCCAG 65° for 1′20″TCAGCCATGGAC GCCCCTACAGGT 72° C. for 1′20″ ATGCTTC (121); AGCTG (122);EcoRI BamHI SST4 Genomic DNA 94° C. for 1′ GCCGAATTCAGC GAGGGATCCACG65° for 1′20″ TGCCCTGCGCCG CAGGGTGGGTAG 72° C. for 1′20″ GCACCCC (123);GGGAAGG (124); EcoRI BamHI SST5 Genomic DNA 94° C. for 1′ TCTAAGCTTGCACCTGAATTCCTGG 65° for 1′20″ GAGCCTGACGCA GGGTGACACGGG 72° C. for 1′20″CCCCAG (125); GCCGCC (126); HindIII EcoRI TSHR Genomic DNA 94° C. for 1′GGCGAATTCGGA GTAGGATCCCCT 65° for 1′ GGATGGAGAAAT ACCATTGTGAGT 72° C.for 2′30″ AGCCCC (127); AGTGTA (128); EcoRI BamHI VIPR1 Lung cDNA 94° C.for 1′ CCGAAGCTTCAG TGGGAATTCGAC 65° for 1′ GGCAGACCATGC CAGGGAGACTTC72° C. for 1′30″ GCCCGCCA (129); GGCTTGGAA HindIII (130); EcoRI VIPR2Brain cDNA 94° C. for 1′ GCTAAGCTTGCC GTGGAATTCGAT 65° for 1′ATGCGGACGCTG GACCGAGGTCTC 72° C. for 1′30″ CTGCCTCCCGCG CGTTTGCAG (132);(131); HindIII EcoRI

B. Expression by Alternative Approaches

1. AVPR1A

The endogenous human AVPR1A was obtained by PCR using a template andrTth polymerase (Perkin Elmer) with the buffer system provided by themanufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides.The cycle condition was 30 cycles of 94° C. for 1 min, 64° C. for 1 minand 72° C. for 1 min and 30 sec. The 5′ PCR fragment was obtainedutilizing genomic DNA, as a template, and the following primer set:

(SEQ.ID.NO.:133) 5′-ATCACCATGCGTCTCTCCGCCGGTCCCGA-3′ and(SEQ.ID.NO.:134) 5′-TTGTTCACCTCGATCATGGAGAAGA-3′.

The 3′ PCR fragment was obtained by pfu polymerase (Stratagene) usingIMAGE 1055179, as a template, and the following primer set:

(SEQ.ID.NO.:135) 5′-CGCAGTACTTCGTCTTCTCCATGA-3′ and (SEQ.ID.NO.:136)5′-CAAGAATTCAGTTGAAACAGGAATGAATTTGATGG-3′.

The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94°C. for 1 min, 60° C. and 72° C. for 1 min 30 sec. The 5′ and 3′ PCRfragments were then used as co-templates to obtain the full length cDNAusing the pfu polymerase and SEQ. ID. NO.:133 and SEQ. ID. NO.:136 asprimers. The cycle condition for each PCR reaction was 30 cycles of 94°C. for 1 min, 65° C. and 72° C. for 2 min 10 sec.

The resulting PCR fragment was digested with EcoRI restriction site andcloned into an EcoRI pCMV expression vector. Nucleic acid and amino acidsequences were thereafter determined and verified.

2. AVPR1A Variant

The endogenous human AVPR1A variant was obtained by PCR using a templateand rTth polymerase (Perkin Elmer) with the buffer system provided bythe manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 64°C. for 1 min and 72° C. for 1 min and 30 sec. The 5′ PCR fragment wasobtained utilizing genomic DNA, as a template, and: SEQ. ID. NO.:133 andSEQ. ID. NO.:134 as primers.

The 3′ PCR fragment was obtained by pfu polymerase (Stratagene) usingIMAGE 1542469, as a template, and SEQ. ID. NO.:136 and5′-ACAGAATTCTCCAGTTCTCATTTTCTTATCCGTAC-3′ (SEQ. ID. NO.:137).

The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94°C. for 1 min, 65° C. for 1 min and 72° C. for 1 min 30 sec. The 5′ and3′ PCR fragments were then used as co-templates to obtain the fulllength cDNA using the pfu polymerase (Stratagene) and SEQ. ID. NO.:133and SEQ. ID. NO.:136 as primers. The cycle condition for each PCRreaction was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C.for 2 min 10 sec.

The resulting PCR fragment was digested with EcoRI restriction site andcloned into an EcoRI pCMV expression vector. Nucleic acid and amino acidsequences were thereafter determined and verified. 3. AVPR1B

The endogenous human AVPR1B was obtained by PCR using a template andrTth polymerase (Perkin Elmer) with the buffer system provided by themanufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides.Both rounds of PCR had the following cycle condition: 30 cycles of 94°C. for 1 min, 65° C. for 1 min and 72° C. for 2 sec. The first round ofPCR utilized a pituitary DNA, as a template, and a 5′ PCR primer thatcontained a HindIII site with the following sequence:5′-GCAAAGCTTGCTCATGGATTCTGGGCCTCT-3′ (SEQ. ID. NO.:138) The 3′ PCRprimer contained an EcoRI site with the following sequence:5′-TCTGAATTCAAAGATGATGGTCTCAGCGGTGCC-3′-(SEQ. ID. NO.:139). The secondround of PCR utilized pituitary DNA as a template and a 5′ PCR primercontained a HindIII site with the following sequence:5′-GCAAAGCTTGCTCATGGATTCTGGGCCTCTGTGGG-3′ (SEQ. ID. NO.:140) and the 3′PCR primer contained an EcoRI site with the following sequence:5′-TCTGAATTCAAAGATGATGGTCTCAGCGGTGCCTTCCC-3′ (SEQ. ID. NO.:141).

The resulting PCR fragment was digested with HindIII and EcoRIrestriction site and cloned into a HindIII-EcoRI pCMN expression vector.Nucleic acid and amino acid sequences were thereafter determined andverified.

4. 5HT6

The endogenous human 5HT6 receptor was obtained by PCR using a templateand rTth polymerase (Perkin Elmer) with the buffer system provided bythe manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4nucleotides. Both rounds of PCR had the following cycle condition: 30cycles of 94° C. for 1 min, 60° C. for 1 min and 72° C. for 1 min and 45sec. The first round of PCR utilized a caudate nucleus DNA, as atemplate, and a 5′ PCR primer that contained a HindIII site with thefollowing sequence: 5′-CATAAGCTTTCCCGCCACCCTATCACT-3′ (SEQ. ID. NO.:142)The 3′ PCR primer contained an EcoRI site with the following sequence:5′-ACTGAATTCTGCTCAATCCAGCTCCCCA-3′-(SEQ. ID. NO.:143). The second roundof PCR also utilized caudate nucleus DNA as a template and a 5′ PCRprimer that contained an EcoRV site with the following sequence:5′-CCTCGGATATCATGGTCCCAGAGCCGGGCCC-3′ (SEQ. ID. NO.:144) and a 3′ PCRprimer that contained a XbaI site with the following sequence:5′-CAGCTCTAGATTGGCCAGCCCCAAGCCCGGGT-3′ (SEQ. ID. NO.:145).

Nucleic acid and amino acid sequences were thereafter determined andverified.

5. Dopamine D1

Dopamine D1 was subcloned from a full length cDNA clone obtained fromthe American Type Culture Collection.

6. Dopamine D2

Dopamine D2 was subcloned from a full length cDNA clone obtained fromthe American Type Culture Collection.

7. Dopamine D5

To obtain Dopamine D5, PCR was performed using genomic cDNA as templateand rTth polymerase (Perkin Elmer) with the buffer system provided bythe manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4nucleotides.

Dopamine D5 receptor contained no intron in the coding region. However,Dopamine D5 receptor contained two pseudogenes with 8 bp fame shiftinsertion within the coding region. In order to avoid the pseudogenes,the DNA fragment 5′ and 3′ of the frame shift insert was each amplifiedfrom genomic DNA. The 5′ PCR fragment was obtained utilizing thefollowing primer set:

(SEQ.ID.NO.:146; sense)     5′-CCTGAATTCCAGCCCGAAATGCTGCCGCCAG-3′(SEQ.ID.NO.:147; antisense) 5′-GGTCCACGCTGATGACGCACAGGTTC-3′

3′ fragment was obtained utilizing the following primer set:

(SEQ.ID.NO.: 148; sense)     5′-GAACCTGTGCGTCATCAGCGTGGACC-3′(SEQ.ID.NO.:149; antisense) 5′-TGCGGATCCATGAGGGGGTTTCTTAATG-3′.

The 5′ and 3′ PCR fragments were then used as co-templates to obtain thefull length cDNA using the pfu polymerase and SEQ. ID. NO.:146 and SEQ.ID. NO.:149 as primers. The cycle condition for each PCR reaction was 30cycles of 94° C. for 1 min, 65° C. for 2 min 30 sec and 72° C. for 1 min30 sec.

The resulting PCR fragment was digested with EcoRI and Bamboorestriction sites and cloned into an EcoRI-BamHI pCMV expression vector.Nucleic acid and amino acid sequences were thereafter determined andverified.

8. ETA

The endogenous human ETA was obtained by PCR using a template and rTthpolymerase (Perkin Elmer) with the buffer system provided by themanufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides.The cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 minand 72° C. for 2 sec. The 5′ PCR fragment, containing a HindIII site,was obtained utilizing genomic DNA, as a template, and the followingprimer set:

(SEQ.ID.NO.:150) 5′-CGGAAGCTTCTGGAGCAGGTAGCAGCATG-3′ and(SEQ.ID.NO.:151) 5′-TGGGCAATAGTTGTGCATTGAGCCA-3′.

The 3′ PCR fragment, containing BamHI site, was obtained by pfupolymerase (Stratagene) using IMAGE 666747, as a template, and thefollowing primer set:

(SEQ.ID.NO.:152) 5′-CTAATTTGGTCCTACCCAGCAATGGC-3′ and (SEQ.ID.NO.:153)5′-CTTGGATCCAGATGAGCTGTATTTATTACTGGAACG-3′.

The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94°C. for 1 min, 64° C. for 1 min and 72° C. for 2 sec. The 5′ and 3′ PCRfragments were then used as co-templates to obtain the full length cDNAusing the pfu polymerase (Stratagene) and the following primers:

(SEQ ID.NO.:154) 5′-AATAAGCTTCAAGATGGAAACCCTTTGCCTCAG-3′(SEQ.ID.NO.:155) 5′-CGTTCATGCTGTCCTTATGGCTGCTC-3′.

The PCR cycle condition for the full length clone was 30 cycles of 94°C. for 1 min, 65° C. for 1 min and 72° C. for 2 min 10 sec.

The resulting PCR fragment was digested with EcoRI restriction site andcloned into an EcoRI pCMV expression vector. Nucleic acid and amino acidsequences were thereafter determined and verified.

9. mGluR1

The endogenous human mGluR1 was obtained by PCR using a template andrTth polymerase (Perkin Elmer) with the buffer system provided by themanufacturer. The cycle condition for the first round of PCR was asfollows: 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for1 min and 50 sec. The 5′ PCR fragment contained a SalI site and wasobtained utilizing hippocampus DNA as a template, and the followingprimer set:

(SEQ.ID.NO.:156) 5′-GCAGGCTGTCGACCTCGTCCTCACCACCATGGTC-3′ and(SEQ.ID.NO.:157) 5′-AATGGGCTCACAGCCTGTTAGATCTGCATTGGGCCAC-3′.

The middle PCR fragment was obtained utilizing genomic DNA as atemplate, where the cycle condition was 30 cycles of 94° C. for 1 min,65° C. for 1 min and 72° C. for 1 min and 50 sec, with the followingprimer set:

(SEQ.ID.NO.:158) 5′-TAACAGGCTGTGAGCCCATTCCTGTGCG-3′ and (SEQ.ID.NO.:159)5′-TTAGAATTCGCATTCCCTGCCCCTGCCTTCTTTC-3′.

The 3′ PCR fragment contained a BamHI site and was obtained utilizinggenomic cDNA, as a template, and the following primer set:

(SEQ.ID.NO.:160) 5′-TGCGAATTCTAATGGCAAGTCTGTGTCATGGTC-3′ and(SEQ.ID.NO.:161) 5′-TCCGGATCCCAGGGTGGAAGAGCTTTGCTTGTA-3′.

The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94°C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 15 sec.

The resulting PCR fragment was digested with SalI and BamHI restrictionsite and cloned into a SalI-BamHI pCMV expression vector. Nucleic acidand amino acid sequences were thereafter determined and verified.

10. GPR24 (Also Known as MCH or SLC-1)

The endogenous human GPR24 was obtained by PCR using genomic DNA astemplate and rTth polymerase (Perkin Elmer) with the buffer systemprovided by the manufacturer, 0.25 μM of each primer, and 0.2 mM of each4 nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min,56° C. for 1 min and 72° C. for 1 min and 20 sec.

The 5′ PCR primer contained a HindIII site with the sequence:5′-GTGAAGCTTGCCTCTGGTGCCTGCAGGAGG-3′ (SEQ. ID. NO.:162) and the 3′primer contained an EcoRI site with the sequence:5′-GCAGAATTCCCGGTGGCGTGTTGTGGTGCCC-3′ (SEQ. ID. NO.:163).

The 1.3 kb PCR fragment was digested with HindIII and EcoRI and clonedinto HindIII-EcoRI site of CMVp expression vector. Later the cloningwork by Lakaye et al showed that there is an intron in the coding regionof the gene. Thus the 5′ end of the cDNA was obtained by 5′ RACE PCRusing Clontech's marathon-ready hypothalamus cDNA as template and themanufacturer's recommended protocol for cycling condition. The 5′ RACEPCR for the first and second round PCR were as follows:

5′-CATGAGCTGGTGGATCATGAAGGG-3′ (SEQ.ID.NO.:164) and5′-ATGAAGGGCATGCCCAGGAGAAAG-3′. (SEQ.ID.NO.:165)

Nucleic acid and amino acid sequences were thereafter determined andverified.

11. NTSR1

The endogenous human NTSR1 was obtained by PCR using a template and rTthpolymerase (Perkin Elmer) with the buffer system provided by themanufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides.The cycle condition was 30 cycles of 94° C. for 1 min, 60° C. for 1 minand 72° C. for 1 min and 10 sec. The 5′ PCR fragment, containing aHindIII site, was obtained utilizing genomic DNA, as a template, and thefollowing primer set:

(SEQ.ID.NO.:166) 5′-CCCAAGCTTCCAGCCCCGGAGGCGCCGGAC-3′ and(SEQ.ID.NO.:167) 5′-TGAAGGTGTTGACCTGTATGACGACCTTGACGGTGGG-3′.

The 3′ PCR fragment, containing an EcoRI site, was obtained utilizingbrain cDNA, as a template, and the following primer set:

(SEQ.ID.NO.:168) 5′-GGTCGTCATACAGGTCAACACCTTCATGTCCTTCATA-3′ and(SEQ.ID.NO.:169) 5′-CACGAATTCGTACAGCGTCTCGCGGGTGGCATT-3′.

The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94°C. for 1 min, 60° C. for 1 min and 72° C. for 1 min and 10 sec. The 5′and 3′ PCR fragments were then used as co-templates to obtain the fulllength cDNA using the pfu polymerase (Stratagene) and5′-ATCACCATGCGCCTCAACAGCTCCGC-3′ (SEQ. ID. NO.:170) and SEQ. ID. NO.:169as primers. The cycle condition for each PCR reaction was 30 cycles of94° C. for 1 min, 65° C. for 1 min and 72° C. for 2 min 10 sec.

The resulting PCR fragment was digested with HindIII and EcoRIrestriction site and cloned into a HindIII-EcoRI pCMV expression vector.Nucleic acid and amino acid sequences were thereafter determined andverified.

12. NTSR2

The endogenous human NTSR2 was obtained by PCR using a template and pfupolymerase (Stratagene) with the buffer system provided by themanufacturer. The cycle condition was 30 cycles of 94° C. for 1 min, 65°C. for 1 min and 72° C. for 1 min and 15 sec. The 5′ PCR fragment wasobtained utilizing IMAGE 1537523, as a template, and the followingprimer set:

(SEQ.ID.NO.:171) 5′-ATCACCATGGAAACCAGCAGCCCGCGGC-3′ and (SEQ.ID.NO.:172)5′-CGGGGTAGAAGTGGACGGCACTTGGG-3′.

The 3′ PCR fragment, containing an EcoRI site, was obtained utilizingcaudate nucleus cDNA, as a template, and the following primer set:

(SEQ.ID.NO.:173) 5′-GCTCCCAAGTGCCGTCCACTTCTACC-3′ and (SEQ.ID.NO.:174)5′-TTAGAATTCGGTCCGGGTTTCTGGGGGATCC-3′.

The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94°C. for 1 min, 60° C. for 1 min and 72° C. for 1 min and 20 sec. The 5′and 3′ PCR fragments were then used as co-templates to obtain the fulllength cDNA using the pfu polymerase (Stratagene) and SEQ. ID. NO.:171and SEQ. ID. NO.:174 as primers. The PCR cycle condition for the fulllength clone was 30 cycles of 94° C. for 1 min, 60° C. for 1 min and 72°C. for 2 min 10 sec.

The resulting PCR fragment was digested with EcoRI restriction site andcloned into an EcoRI pCMV expression vector. Nucleic acid and amino acidsequences were thereafter determined and verified.

13. OPRM1

The endogenous human OPRM1 was obtained by PCR using a template and rTthpolymerase (Perkin Elmer) with the buffer system provided by themanufacturer. The cycle condition was 30 cycles of 94° C. for 1 min, 65°C. for 1 min and 72° C. for 40 sec. The 5′ PCR fragment, containing aHindIII site, was obtained utilizing genomic DNA as a template, and thefollowing primer set:

(SEQ.ID.NO.:175) 5′-CCCAAGCTTCAGTACCATGGACAGCAGCGCTGCC-3′ and(SEQ.ID.NO.:176) 5′-CATCTTGGTGTATCTGACAATCACATACATGACCAGGAA-3′.

The middle PCR fragment was obtained utilizing genomic DNA as atemplate, where the cycle condition was 30 cycles of 94° C. for 1 min,60° C. for 1 min and 72° C. for 40 sec, and the following primer set:

(SEQ.ID.NO.:177) 5′-GTATGTGATTGTCAGATACACCAAGATGAAGACTGCCAC-3′ and(SEQ.ID.NO.:178) 5′-TACAATCTATGGAACCTTGCCTGTATTTTGTTGTAGCCA-3′.

The 3′ PCR fragment was obtained utilizing brain cDNA, as a template,and the following primer set:

(SEQ.ID.NO.:179) 5′-CAAAATACAGGCAAGGTTCCATAGATTGTACACTAACAT-3′ and(SEQ.ID.NO.:180) 5′-CGGGCAACGGAGCAGTTTCTGCTTCAG-3′.

The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94°C. for 1 min, 63° C. for 1 min and 72° C. for 1 min and 15 sec.

The 5′PCR fragment and the middle PCR fragment were used as templates toobtain the 5′-region through the middle region of OPRM1 (“5′-middle PCRfragment”) using the pfu polymerase (Stratagene) and SEQ. ID. NO.:175and SEQ. ID. NO.:178 with the cycle conditions as follows: 30 cycles of94° C. for 1 min, 63° C. for 1 min and 72° C. for 1 min and 15 sec.

The 5′-middle PCR fragment and 3′ PCR fragment were then used astemplates to obtain the full length cDNA using the pfu polymerase(Stratagene) and SEQ. ID. NO.:175 and SEQ. ID. NO.:180 as primers. Thecycle condition for the full length PCR reaction was 30 cycles of 94° C.for 1 min, 65° C. for 1 min and 72° C. for 1 min 15 sec.

The resulting PCR fragment was digested with Hindi restriction site andcloned into a HindIII pCMV expression vector. Nucleic acid and aminoacid sequences were thereafter determined and verified.

14. OPRM1A

The endogenous human OPRM1A was obtained by PCR using a template andrTth polymerase (Perkin Elmer) with the buffer system provided by themanufacturer. The cycle condition was 30 cycles of 94° C. for 1 min, 65°C. for 1 min and 72° C. for 40 sec. The 5′ PCR fragment, containing aHindIII site, was obtained utilizing genomic DNA as a template, and SEQ.ID. NO.:175 and SEQ. ID. NO.:176.

The middle PCR fragment was obtained utilizing genomic DNA as atemplate, where the cycle condition was 30 cycles of 94° C. for 1 min,60° C. for 1 min and 72° C. for 40 sec, and SEQ. ID. NO.:177 and SEQ.ID. NO.:178.

The 3′ PCR fragment was obtained utilizing genomic DNA as a template,and SEQ. ID. NO.:179 and SEQ. ID. NO.:180. The cycle condition for 3′PCR reaction was as follows: 30 cycles of 94° C. for 1 min, 60° C. for 1min and 72° C. for 1 min and 30 sec.

The 5′PCR fragment and the middle PCR fragment were used as co-templatesto obtain the 5′-region through the middle region of OPRM1A (“5′-middlePCR fragment”) using the pfu polymerase (Stratagene) and SEQ. ID.NO.:175 and SEQ. ID. NO.:178 with the cycle conditions as follows: 30cycles of 94° C. for 1 min, 63° C. for 1 min and 72° C. for 1 min and 15sec.

The 5′-middle PCR fragment and 3′ PCR fragment were then used asco-templates to obtain the full length cDNA using the pfu polymerase(Stratagene) and SEQ. ID. NO.:175 and SEQ. ID. NO.:180 as primers. Thecycle condition for the full length PCR reaction was 30 cycles of 94° C.for 1 min, 65° C. for 1 min and 72° C. for 1 min 15 sec.

The resulting PCR fragment was digested with HindIII restriction siteand cloned into a HindIII pCMV expression vector. Nucleic acid and aminoacid sequences were thereafter determined and verified.

15. OX₁R

The OX₁R EST clone 40608 is a full length cDNA clone. However, itcontained a 4 bp frame shift insertion. To remove the insert, thefragments 5, and 3, of the frame shift insert was each obtained by PCRusing EST clone 40608 as template and two primer pairs. The 5′ primerset, containing an EcoRI site, were as follows:

(SEQ.ID.No.:181; sense)     5′-ATGGAATTCTGCTGCAGCGGCTCCTGAGCTC-3′(SEQ.ID.No.:182; antisense) 5′-ACGGACACAGCCTGTAGATAGGGGATGACCTTGCAG-3′

and the 3′ primer set, containing a BamHI site, were as follows:

(SEQ.ID.NO.:183; sense)     5′-ATCCCCTATCTACAGGCTGTGTCCGTGTCAGTGGCAG-3′(SEQ.ID.NO.:184; antisense) 5′-GGAGGATCCAGGGCAGCCCTCGCTCAGGGC-3′.

The 5′ and 3, PCR fragments were then used as cotemplates to obtain thefull length cDNA using SEQ. ID. NO.:181 and SEQ. ID. NO.:184 as primers.The cycle condition for each PCR reaction was 30 cycles of 94° C. for 1min, 65° C. for 2 min 30 sec and 72° C. for 1 min 30 sec.

The resulting PCR fragment was digested with EcoRI and BamHI restrictionsites and cloned into an EcoRI-BamHI pCMV expression vector. Nucleicacid and amino acid sequences were thereafter determined and verified.

16. PTHR1

The endogenous human PTHR1 was obtained by PCR using a template and rTthpolymerase (Perkin Elmer) with the buffer system provided by themanufacturer. The cycle condition was 30 cycles of 94° C. for 1 min, 65°C. for 1 min and 72° C. for 1 min and 30 sec. The 5′ PCR fragment,containing a HindIII site, was obtained utilizing kidney cDNA, as atemplate, and the following primer set:

(SEQ.ID.NO.:185) 5′-CGCAAGCTTAGGCGGTGGCGATGGGGACCGCC-3′ and(SEQ.ID.NO.:186) 5′-GGATGTGGTCCCATTCCGGCAGACAG-3′.

The 3′ PCR fragment, containing an EcoRI site, was obtained by pfu PCR(Stratagene) and IMAGE 1624048, as a template, and the following primerset:

(SEQ.ID.NO.:187) 5′-AGGAGGCACCCACTGGCAGCAGGTA-3′ and (SEQ.ID.NO.:188)5′-GCCGAATTCCATGACTGTCTCCCACTCTTCCTG-3′.

The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94°C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 30 sec. The 5′and 3′ PCR fragments were then used as co-templates to obtain the fulllength cDNA using the pfu polymerase (Stratagene) and SEQ. ID. NO.:185and SEQ. ID. NO.:188 as primers. The PCR cycle condition for the fulllength clone was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72°C. for 3 sec.

The resulting PCR fragment was digested with HindIII and EcoRIrestriction site and cloned into a HindIII-EcoRI pCMV expression vector.Nucleic acid and amino acid sequences were thereafter determined andverified.

17. SST2

SST2 was obtained by subcloning EST 06818 into a pCMN vector.

Table E below indicates the GenBank Accession number for which theendogenous receptors set forth above can be located, and for which theendogenous nucleic and amino acid sequences are provided.

TABLE E Receptor Identifier GenBank Accession Number 5HT-1A X135565HT-1B D10995 5HT-1D M81589 5HT-1E M91467 5HT-1F L04962 5HT-2B X773075HT-4A Y08756 5HT-4B Y12505 5HT-4C Y12506 5HT-4D Y12507 5HT-4E AJ0113715HT-5A X81411 5HT6 L41147 5HT7 L21195 AVPR1A AF030625 AVPR1B D31833AVPR2 NM_000054 BBR3 X76498 BDKR1 AJ238044 BDKR2 NM_000623 C3a U62027C5a M62505 CB1 X54937 CB2 X74328 CCR2b U03882 CCR3 U28694 CCR5 U54994CCR8 U45983 CCR9 AJ132337 CRFR1 L23332 CXCR4 AJ224869 Dopamine D1 X55758Dopamine D2 S62137 Dopamine D3 U32499 Dopamine D5 M67439 ETA X61950 ETBL06623 FPR1 M60627 FPRL1 M76672 GALR1 L34339 GALR2 AF040630 GALR3AF073799 GIP U39231 mGluR1 L76627 GPR5 L36149 GPR24 (also known as MCHor SLC-1) U71092 GRPR M73481 M1 X15263 M2 X15264 M3 X15266 M4 X15265 M5M80333 MC3 L06155 NK1R M74290 NK2R M57414 NK3R M89473 NMBR M73482 NPY5U94320 NTSR1 X70070 NTSR2 Y10148 OPRD U07882 OPRL1 X77130 OPRK U11053OPRM L25119 OPRM1A L25119 OX₁R AF041243 OX₂R AF041245 PACAP D17516 PAFS56396 PGE EP1 L22647 PGE EP2 U19487 PGE EP4 NM_000958 PTHR1 L04308PTHR2 U25128 SCTR U28281 SST1 M81829 SST2 M81830 SST3 M96738 SST4 D16826SST5 D16827 TSHR AF035261 VIPR L13288 VIPR2 X95097

Example 2

Preparation of Non-Endogenous, Versions of the Known GPCRS

A. Site-Directed Mutagenesis

Those skilled in the art are credited with the ability to selecttechniques for mutation of a nucleic acid sequence. Presented below areapproaches utilized to create non-endogenous versions of several of thehuman GPCRs disclosed above. The mutations disclosed below are basedupon an algorithmic approach whereby the 16^(th) amino acid (located inthe IC3 region of the GPCR) from a conserved proline residue (located inthe TM6 region of the GPCR, near the TM6/IC3 interface) is mutated, mostpreferably to a lysine amino acid residue.

In most of the examples of this Example 2, the algorithmic approach setforth above was used to identify the amino acid residue to be mutated.However, several GPCRs set forth below utilized a modified algorithmicapproach (e.g., CRFR1, GIP, mGluR1, GPR24, PTHR1, PTHR2, SCTR, TSHR,VIPR and VIPR2). This modified approach focuses on a conserved prolineresidue (also located in the TM6 region of the GPCR, near the TM6/IC3interface) whereby the 5^(th) amino acid upstream from the proline isgenerally, but not always, a threonine residue. For these receptors, theendogenous 5^(th) amino acid residue is mutated, most preferably to aproline amino acid residue.

Other mutation approaches can be used (e.g., mGluR1, GPR24 and TSHR) andone skilled in the art is credited with the ability to select techniquesfor mutation of a nucleic acid sequence. The importance here is that themutation leads to a constitutively activated receptor and given theextension approaches set forth herein for determination ofconstitutively activity, routine analysis can be employed in thiscontext.

Preparation of non-endogenous known GPCRs is preferably accomplished byusing TRANSFORMER SITE-DIRECTED™ Mutagenesis Kit (Stratagene, accordingto manufacturer's instructions) or QUIKCHANGE SITE-DIRECTED™ Mutagenesis(Clontech). Endogenous GPCR is preferably used as a template and twomutagenesis primers utilized, as well as, most preferably, a lysinemutagenesis oligonucleotide and a selection marker oligonucleotide (SEQ.ID. NO.:252; included in Stratagene's kit). For convenience, the codonmutation incorporated into the known GPCR and the respectiveoligonucleotides are noted, in standard form (Table F):

TABLE F 5′-3′ orientation (sense), Receptor Codon (SEQ.ID.NO.) mutation5′-3′ orientation (antisense) Identifier Mutation underlined(SEQ.ID.NO.) 5HT-1A V343K CGAGAGAGGAAGACAAAG ATGCCCAGCGTCTTCTTTAAGACGCTGGGCAT (189) GTCTTCCTCTCTCG (190) 5HT-1B T313KGGGAGCGCAAAGCCAAGA GATCCCTAGGGTCTTCTT AGACCCTAGGGATC (191)GGCTTTGCGCTCCC (192) 5HT-1D T300K CGAGAAAGGAAAGCCAAG GAATGATGCCCAGGATTAAAATCCTGGGCATCATTC TTCTTGGCTTTCCTTTCT (193) CG(194) 5HT-1E A290KAGGGAACGGAAGGCAAAA AGCCCCAGGATGCGTTT CGCATCCTGGGGCT (195)TGCCTTCCGTTCCCT (196) 5HT-1F A292K CAAGAGAACGGAAAGCAA GATTAATCCCAGGGTAGAGACTACCCTGGGATTAAT TCTTTGCTTTCCGTTCTC C(197) TTG(198) 5HT-2B S323KAACGAACAGAGAGCCAAA CAATCCCTAGGACCTTTT AAGGTCCTAGGGATTG TGGCTCTCTGTTCGTT(199) (200) 5HT-4A A258K GGACAGAGACCAAAGCAA GATGCACAGGGTCTTCTTAGAAGACCCTGTGCATC TGCTTTGGTCTCTGTCC (201) (202) 5HT-4B A258KGGACAGAGACCAAAGCAA GATGCACAGGGTCTTCTT AGAAGACCCTGTGCATCTGCTTTGGTCTCTGTCC (203) (204) 5HT-4C A258K GGACAGAGACCAAAGCAAGATGCACAGGGTCTTCTT AGAAGACCCTGTGCATC TGCTTTGGTCTCTGTCC (205) (206)5HT-4D A258K GGACAGAGACCAAAGCAA GATGCACAGGGTCTTCTT AGAAGACCCTGTGCATCTGCTTTGGTCTCTGTCC (207) (208) 5HT-4E A258K GGACAGAGACCAAAGCAAGATGCACAGGGTCTTCTT AGAAGACCCTGTGCATC TGCTTTGGTCTCTGTCC (209) (210)5HT-5A A284K AAGGAGCAGCGGGCCAAG GATGCCCACCATGAGCT CTCATGGTGGGCATC (211)TGGCCCGCTGCTCCTT (212) 5HT-6 S267K CTGAAGGCCAAGCTTACGCTATGCCCAGCGTAAGCTTG GGGCATCCTGCTGGGCA GCCTTCAGGGCCTTCCTG (213) CT (214)5HT-7 A326K GAACAGAAAGCAAAGACCA CCCAGGGTGGTCTTTGCT CCCTGGGGATCATCGT(215) TTCTGTTCTCGCTTAAA (216) AVPR1A V290K GCCAAGATCCGCACGAAGACGATCACAAAAGTCATCT AGATGACTTTTGTGATCG TCTTCGTGCGGATCTTGG (217) C(218)AVPR1B V280K GGCCAAGATCCGAACAAAG CGATGACAAAGGTCATCT AAGATGACCTTTGTCATCTCTTTGTTCGGATCTTGG (219) CC (220) AVPR2 V270K GCTGTGGCCAAGACTAAGACACTAGCGTCATCCTCTT GGATGACGCTAGTG (221) AGTCTTGGCCACAGC (222) BBR3 270KCGAAAGAGAATTAAAAGAA AATACCGTTCTTTTAATT CGGTATTGGTGTTG (223)CTCTTTCGGGATTC (224) BDKR1 T249K GCCGCAAGGATAGCAAGAC GTGAGGATCAGCGCTTTGCAAAGCGCTGATCCTCAC GTCTTGCTATCCTTGCGG (225) C (226) BDKR2 T269KCGGAGAGGAGGGCCAAGGT ACCAGGACTAGCACCTTG GCTAGTCCTGGT (227) GCCCTCCTCTCCG(228) C3a F376K CGCCAAGTCTCAGAGCAAA CACCACGGCCACTCGCTTACCAAGCGAGTGGCCGTGG GGTTTTGCTCTGAGACTT TG (229) GGCG (230) C5a L241KCGGTCCACCAAGACAAAGA TGCCACCACCACCTTCTT AGGTGGTGGTGGCA (231)TGTCTTGGTGGACCG (232) CB1 A342K CGCATGGACATTAGGTTAA TCAGGACCAGGGTCTTCTAGAAGACCCTGGTCCTGA TTAACCTAATGTCCATGC (233) G (234) CB2 A244KGGCTGGATGTGAGGTTGAA CACTAGCCCTAGGGTCTT GAAGACCCTAGGGCTAGTGCTTCAACCTCACATCCAG (235) CC (236) CCR2b V242K GAAGAGGCATAGGGCAAAGGGTGAAGATGACTCTCTT AGAGTCATCTTCACC (237) TGCCCTATGCCTCTTC (238) CCR3I238K GTAAAAAAAAGTACAAGGC GATGACAAAAATGAGCC CAAGCGGCTCATTTTTGTCAGCTTGGCCTTGTACTTTTT TC (239) TTTAC (240) CCR5 V234K GAAGAGGCACAGGGCTAAGGATGGTGAAGATAAGCC AGGCTTATCTTCACCATC TCTTAGCCCTGTGCCTCT (241) TC (242)CCR8 I237K CCACAACAAGACCAAGGCC CCACAATGAGCACCAAC AAGAGGTTGGTGCTCATTGTCTCTTGGCCTTGGTCTTG GG (243) TTGTGG (244) CCR9 L253K TCCAAGCACAAAGCCAAAAGGACAGTGATGGTCACTT AAGTGACCATCACTGTCC TTTTGGCTTTGTGCTTGG (245) A (246)CRFR1 T316P GAAGGCTGTGAAAGCCCCT GCAGCAGCACCAGAGGG CTGGTGCTGCTGC (247)GCTTTCACAGCCTTC (248) CXCR4 L238K AGAAGCGCAAGGCCAAGAA TGAGGATGACTGTGGTCTGACCACAGTCATCCTCA TCTTGGCCTTGCGCTTCT (249) (250) Dopamine D1 L271KGAGAAACTAAAGTCAAGAA CTCCTTCGGTCCTCCTAT GACTCTGTG (251) CGTTGTCAGAAGT(252) Dopamine D2 T372K GAGAAGAAAGCCAATCAGA GGCGAGCATCTGAGTGG TGCTCGCC(253) CTTTCTTCTC (254) Dopamine D3 T328K GAGAAGAAGGCAAAACAAAGGCCACCATTTGTTTTGC TGGTGGCC (255) CTTCTTCTC (256) Dopamine D5 L295KAAGAAGGAGACCAAAGTTA CGACAGGGTCTTTTTAAC AAAAGACCCTGTCG (257)TTTGGTCTCCTTCTT (258) ETA A305K CAGCGTCGAGAAGTGAAAA TACAACCAAGCAGAAAAAAACAGTTTTCTGCTTGGTT CTGTTTTTTTCACTTCTCG GTA (259) ACGCTG (260) ETBA322K CAGAGACGGGAAGTGAAGA CCAGGCAAAAGACGGTT AAACCGTCTTTTGCCTGGTTCTTCACTTCCCGTCTCT (261) G(262) FPR1 L240K AAGTCCAGTCGTCCCAAACAAGGAGAGGACCCGTTT GGGTCCTCTCCTT (263) GGGACGACTGGACTT (264) FPRL1 L240KAAATCCAGCCGTCCCAAAC GCAGTGAGGACCCGTTTG GGGTCCTCACTGC (265)GGACGGCTGGATTT (266) GALR1 A246K CCAAGAAAAAGACTAAACA CTCCTTCGGTCCTCCTATGACAGTTCTGG (267) CGTTGTCAGAAGT (252) GALR2 T235K GCCAAGCGCAAGGTGAAACGAGGATCATGCGTTTCAC GCATGATCCTC (268) CTTGCGCTTGGCG (269) GIP T343PAGGCTGGCTCGCTCCCCGCT GCACCAGCGTCAGCGGG GACGCTGGTGC (270) GAGCGAGCCAGCCT(271) mGluR1 3′ Deletion See alternative approach below See alternativeapproach below GPR5 V224K CGGCGCCACCGCACGAAAA CTCCTTCGGTCCTCCTATAGCTCATCTTC (272) CGTTGTCAGAAGT (252) GPR24 (also T255K See alternativeapproach below See alternative approach known as MCH T255K/T257R belowor SLC-1) 24-IC3-SST2 C305Y P271L W269C W269F W269L F265I I261Q D140NGRPR A263K GGAAGCGACTTAAGAAGAC CAGCACTGTCTTCTTAAG AGTGCTGGTGTTT (273)TCGCTTCCGGGATTC (274) M1 A364K AAGGAGAAGAAGGCGAAAC GGCACTCAGGGTCCGTTTGGACCCTGAGTGCC (275) CGCCTTCTTCTCCTT (276) M2 T386K CCGGGAAAAGAAAGTCAAGAGCCAAGATTGTCCTCTT AGGACAATCTTGGCT (277) GACTTTCTTTTCCCGG (278) M3 A490KGGTCAAGGAGAAGAAAGCG CGCACTGAGGGTCTGTTT AAACAGACCCTCAGTGCGCGCTTTCTTCTCCTTGACC (279) (280) M4 T399K GGGAGCGCAAAGTGAAACGGGCAAAGATCGTTCGTTT AACGATCTTTGCC (281) CACTTTGCGCTCCC (282) M5 A441KGTCAAAGAGAGGAAAGCAA GGCACTCAGTGTCTGTTT AACAGACACTGAGTGCCTGCTTTCCTCTCTTTGAC (283) (284) MC3 A241K GCAACACTCATGTATGAAGCTCCTTCGGTCCTCCTAT GGGAAAGTCACCATCACC CGTTGTCAGAAGT (252) (285) NK1RV247K GCCAAGCGCAAGGTGAAGA GACAATCATCATTTTCTT AAATGATGATTGTC (286)CACCTTGCGCTTGGC (287) NK2R V249K GCCAAGAAGAAGTTTAAGA AGCACCATGGTCTTCTTAAGACCATGGTGCT (288) AACTTCTTCTTGGC (289) NK3R V298K GGCCAAAAGAAAGGTTAAGCAATAATCATCATTTTCT AAAATGATGATTATTG (290) TAACCTTTCTTTTGGCC (291) NMBRA265K CACGGAAACGCCTGAAAAA CAAGCACAATTTTTTTCA AATTGTGCTTG (292)GGCGTTTCCGTG (293) NPY5 F367K GAATAAAAAAGAGATCACG GGTCAGTCTGTACTTAACAAGTGTTAAGTACAGACTG ACTTCGTGATCTCTTTTTT ACC (294) AT (295) NTSR1 V302KGCCCTGCGGCACGGCAAGC GCACGTAGGACGCGCTTG GCGTCCTACGTGC (296)CCGTGCCGCAGGGC (297) NTSR2 V269K AGCCTCCAGCGCAGCAAGC GGCTCTGAGAACCTGCTTAGGTTCTCAGAGCC (298) GCTGCGCTGGAGGCT (299) OPRD T260KGCCTGCGGCGCATCAAGCG ACCAGCACCATGCGCTTG CATGGTGCTGGT (300) ATGCGCCGCAGGC(301) OPRL1 T262K ACCTGCGGCGCATCAAGCG CACCAGCACCAGCCGCTT GCTGGTGCTGGTG(302) GATGCGCCGCAGGT (303) OPRK T273K ACCTGCGTAGGATCAAGAGCACCAGGACCAGTCTCTT ACTGGTCCTGGTG (304) GATCCTACGCAGGT (305) OPRM T281KGGGAATCTTCGAAGGATCA CACCAGCACCATCCTCTT AGAGGATGGTGCTGGTGGATCCTTCGAAGATTCC (306) (307) OPRM1A T281K GGGAATCTTCGAAGGATCACACCAGCACCATCCTCTT AGAGGATGGTGCTGGTG GATCCTTCGAAGATTCC (308) (309)OX_(I)R A297K CGGAGGAAGACAAAAAAGA CCATCAGCATCTTTTTTG TGCTGATGG (310)TCTTCCTCCG (311) OX₂R A303K CCAGAAGGAAAACAAAACG CTCCTTCGGTCCTCCTATGATGTTGATG (312) CGTTGTCAGAAGT (252) PACAP T355K CGACTGGCCCGGTCCCCCCTGGATGAGCAGCAGGGGG GCTGCTCATCC (313) GACCGGGCCAGTCG (314) PAF L231KGTCAAGCGCCGGGCGAAGT GTGCACACCATCCACTTC GGATGGTGTGCAC (315)GCCCGGCGCTTGAC (316) PGE EP1 V296K CACGACGTGGAGATGAAGGCCGACAAGCTGGCCCTTC GCCAGCTTGTCGG (317) ATCTCCACGTCGTG (318) PGE EP2L263K GAGGAGACGGACCACAAGA CATGATAGCCAGGAGAA TTCTCCTGGCTATCATG (319)TCTTGTGGTCCGTCTCCT C (320) PGE EP4 V271K GCCGAGATCCAGATGAAGAGGTGGCAATGAGTAAGA TCTTACTCATTGCCACC (321) TCTTCATCTGGATCTCGG C (322)PTHR1 T410P GGAAGCTGCTCAAATCCCC GCATGAGCACCAGCGGG GCTGGTGCTCATGC (323)GATTTGAGCAGCTTCC (324) PTHR2 T365P GGAAACTGGCCAAATCGCC GGACCAGGACCAGTGGCACTGGTCCTGGTCC (325) GATTTGGCCAGTTTCC (326) SCTR T344PCGCCTGGCCAGGTCCCCTCT GGATCAGCAGGAGAGGG CCTGCTGATCC (327) GACCTGGCCAGGCG(328) SST1 T270K CGAGCGCAAGATCAAATTA CTCCTTCGGTCCTCCTAT ATGGTGATGG (329)CGTTGTCAGAAGT (252) SST2 T255K AGAAGAAGGTCAAACGAAT GGACACCATTCGTTTGACGGTGTCCATCGTG (330) CTTCTTCTCAGACT (331) SST3 T256K GAACGCAGGGTCAAGCGCACTCCTTCGGTCCTCCTAT TGGTGGTGGCC (332) CGTTGTCAGAAGT (252) SST4 T258KCGGAGAAGAAAATCAAAAG CTCCTTCGGTCCTCCTAT GCTGGTGCTG (333) CGTTGTCAGAAGT(252) SST5 T247K TCGGAGCGAAAGGTGAAGC ACCATGCGCTTCACCTTT GCATGGTGTTGGTGGT(334) CGCTCCGAGCGCCGCCG (335) TSHR V509A See alternative approach belowSee alternative approach D619G below A623I A623K C672Y D619G/A623KV509A/C672Y V509A/A623K/C672Y VIPR T343P AGGCTAGCCAGGTCCCCACTGGATCAGCAGGAGTGGG CCTGCTGATCC (336) GACCTGGCTAGCCT (337) VIPR2 T330PAGGCTGGCCAAGTCCCCGCT GGATAAGCAGGAGCGGG CCTGCTTATCC (338) GACTTGGCCAGCCT(339)

B. Alternative Approaches to Mutation

1. mGluR1

Preparation of a non-endogenous version of the human mGluR1 receptor wasaccomplished by deleting a portion of the intracellular region at the 3′end. The non-endogenous human mGluR1 was obtained by PCR using atemplate and rTth polymerase (Perkin Elmer) with the buffer systemprovided by the manufacturer. The cycle condition for the first round ofPCR was as follows: 30 cycles of 94° C. for 1 min, 65° C. for 1 min and72° C. for 1 min and 50 sec. The 5′ PCR fragment contained a SalI siteand was obtained utilizing hippocampus DNA as a template, and thefollowing primer set:

(SEQ.ID.NO.:340) 5′-GCAGGCTGTCGACCTCGTCCTCACCACCATGGTC-3′ and(SEQ.ID.NO.:341) 5′-AATGGGCTCACAGCCTGTTAGATCTGCATTGGGCCAC-3′.

The middle PCR fragment was obtained utilizing genomic DNA as atemplate, where the cycle condition was 30 cycles of 94° C. for 1 min,65° C. for 1 min and 72° C. for 1 min and 50 sec, with the followingprimer set:

(SEQ.ID.NO.:342) 5′-TAACAGGCTGTGAGCCCATTCCTGTGCG-3′ and (SEQ.ID.NO.:343)5′-TTAGAATTCGCATTCCCTGCCCCTGCCTTCTTTC-3′.

The 3′ PCR fragment was obtained by utilizing the endogenous mGluR1clone as a co-template to obtain the full length cDNA using the pfupolymerase. The cycle condition for this PCR reaction was 30 cycles of94° C. for 1 min, 65° C. for 2 min 30 sec and 72° C. for 1 min 30 sec.and the following primer set:

(SEQ.ID.NO.:344) 5′-TGCGAATTCTAATGGCAAGTCTGTGTCATGGTC-3′ and(SEQ.ID.NO.:345) 5′-TGCGGATCCTCTTCGGAAGATGTTGAGGAAAGTG-3′.

(See, SEQ. ID. NO.:346 for nucleic acid sequence and SEQ. ID. NO.:347for amino acid sequence).

2. GPR24 (MCH or SLC-1)

Preparation of non-endogenous versions of the human GPR24 receptor wasaccomplished by creating an T255K mutation (see, SEQ. ID. NO.:350 fornucleic acid sequence, SEQ. ID. NO.:351 for amino acid sequence), aT255K/T257R mutation (see, SEQ. ID. NO.:354 for nucleic acid sequence,SEQ. ID. NO.:355 for amino acid sequence), an 24-IC3-SST3 mutation (see,SEQ. ID. NO.:358 for nucleic acid sequence, SEQ. ID. NO.:359 for aminoacid sequence), a C305Y mutation (see, SEQ. ID. NO.:362 for nucleic acidsequence, SEQ. ID. NO.:363 for amino acid sequence), a P271L mutation(see, SEQ. ID. NO.:366 for nucleic acid sequence, SEQ. ID. NO.:367 foramino acid sequence), a W269C mutation (see, SEQ. ID. NO.:370 fornucleic acid sequence, SEQ. ID. NO.:371 for amino acid sequence), aW269F mutation see, SEQ. ID. NO.:374 for nucleic acid sequence, SEQ. ID.NO.:375 for amino acid sequence), and a W269L mutation (see, SEQ. ID.NO.:378 for nucleic acid sequence, SEQ. ID. NO.:379 for amino acidsequence), a F265I mutation see, SEQ. ID. NO.:382 for nucleic acidsequence, SEQ. ID. NO.:383 for amino acid sequence), an I261Q mutationsee, SEQ. ID. NO.:386 for nucleic acid sequence, SEQ. ID. NO.:387 foramino acid sequence), and a D140N mutation see, SEQ. ID. NO.:390 fornucleic acid sequence, SEQ. ID. NO.:391 for amino acid sequence).

A. T255K Mutation

Preparation of a non-endogenous version of the human GPR24 receptor wasaccomplished by creating an T255K mutation (see, SEQ. ID. NO.:350 fornucleic acid sequence, and SEQ. ID. NO.:351 for amino acid sequence).Mutagenesis was performed using Transformer Site-Directed MutagenesisKit (Clontech) according to the manufacturer. The PCR sense mutagenesisprimer used had the following sequence:5′-AGAGGGTGAAACGCACAGCCATCGCCATCTG-3′ (SEQ. ID. NO.:348) and theantisense primer (selection marker) had the following sequence:5′-CTCCTTCCGGTCCTCCTATCGTTGTCAGAAGT-3′ (SEQ. ID. NO.:349). Theendogenous GPR24 cDNA was used as a template.

B. T255K/T257R Mutation

Preparation of a non-endogenous version of the human GPR24 receptor wasaccomplished by creating an T255K/T257R mutation (see, SEQ. ID. NO.:354for nucleic acid sequence, and SEQ. ID. NO.:355 for amino acidsequence). Mutagenesis was performed using QuikChange Site-DirectedMutagenesis Kit (Stratagene) according to the manufacturer. The PCRsense mutagenesis primer used had the following sequence:5′-AGAGGGTGAAACGCAGAGCCATCGCCATCTG-3′ (SEQ. ID. NO.:352) and theantisense primer had the following sequence:5′-CAGATGGCGATGGCTCTGCGTTTCACCCTCT-3′ (SEQ. ID. NO.:353). The endogenousGPR24 cDNA was used as a template.

C. 24-IC3-SST2 Mutation

Preparation of a non-endogenous version of the human GPR24 receptor wasaccomplished by creating a 24-IC3-SST2 mutation (see; SEQ. ID. NO.:358for nucleic acid sequence, and SEQ. ID. NO.:359 for amino acidsequence). Blast result showed that GPR24 had the highest sequencehomology to SST2. Thus the IC3 loop of GPR24 was replaced with that ofSST2 to see if the chimera would show constitutive activity.

The BamHI-BstEII fragment containing IC3 of GPR24 was replaced withsynthetic oligonucleotides that contained the IC3 of SST2. The PCR sensemutagenesis primer used had the following sequence:

5′-GATCCTGCAGAAGGTGAAGTCCTCTGGAATCCGAGTGGGCTCCTCTAAGAGGAAGAAGTCTGAGAAGAAG-3′(SEQ.ID.NO.:356)

and the antisense primer had the following sequence:

5′-GTGACCTTCTTCTCAGACTTCTTCCTCTTAGAGGAGCCCACTCGGATTCCAGAGGACTTCACCTTCTGCAG-3′.(SEQ.D.NO.:357)

The endogenous GPR24 cDNA was used as a template.

D. C305Y Mutation

Preparation of a non-endogenous version of the human GPR24 receptor wasaccomplished by creating an C305Y mutation (see, SEQ. ID. NO.:362 fornucleic acid sequence, and SEQ. ID. NO.:363 for amino acid sequence).Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit(Stratagene) according to the manufacturer. The PCR sense mutagenesisprimer used had the following sequence:5′-GGCTATGCCAACAGCTACCTCAACCCCTTTGTG-3′ (SEQ. ID. NO.:360) and theantisense primer had the following sequence:5′-CACAAAGGGGTTGAGGTAGCTGTTGGCATAGCC-3′ (SEQ. ID. NO.:361). Theendogenous GPR24 cDNA was used as a template.

E. P271L Mutation

Preparation of a non-endogenous version of the human GPR24 receptor wasaccomplished by creating an P271L mutation (see, SEQ. ID. NO.:366 fornucleic acid sequence, and SEQ. ID. NO.:367 for amino acid sequence).Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit(Stratagene) according to the manufacturer. The PCR sense mutagenesisprimer used had the following sequence:5′-TTGTGTGCTGGGCACTCTACTATGTGCTACAGC-3′ (SEQ. ID. NO.:364) and theantisense primer had the following sequence:5′-GCTGTAGCACATAGTAGAGTGCCCAGCACACAA-3′ (SEQ. ID. NO.:365). Theendogenous GPR24 cDNA was used as a template.

F. W269C Mutation

Preparation of a non-endogenous version of the human GPR24 receptor wasaccomplished by creating an W269C mutation (see, SEQ. ID. NO.:370 fornucleic acid sequence, and SEQ. ID. NO.:371 for amino acid sequence).Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit(Stratagene) according to the manufacturer. The PCR sense mutagenesisprimer used had the following sequence:5′-GGTCTTCTTTGTGTGCTGCGCACCCTACTATGTG-3′ (SEQ. ID. NO.:368) and theantisense primer had the following sequence:5′-CACATAGTAGGGTGCGCAGCACACAAAGAAGACC-3′ (SEQ. ID. NO.:369). Theendogenous GPR24 cDNA was used as a template.

G. W269F Mutation

Preparation of a non-endogenous version of the human GPR24 receptor wasaccomplished by creating an W269F mutation (see, SEQ. ID. NO.:374 fornucleic acid sequence, and SEQ. ID. NO.:375 for amino acid sequence).Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit(Stratagene) according to the manufacturer. The PCR sense mutagenesisprimer used had the following sequence:5′-GGTCTTCTTTGTGTGCTTCGCACCCTACTATGTG-3′ (SEQ. ID. NO.:372) and theantisense primer had the following sequence:5′-CACATAGTAGGGTGCGAAGCACACAAAGAAGACC-3′ (SEQ. ID. NO.:373). Theendogenous GPR24 cDNA was used as a template.

H. W269L Mutation

Preparation of a non-endogenous version of the human GPR24 receptor wasaccomplished by creating an W269L mutation (see, SEQ. ID. NO.:378 fornucleic acid sequence, and SEQ. ID. NO.:379 for amino acid sequence).Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit(Stratagene) according to the manufacturer. The PCR sense mutagenesisprimer used had the following sequence:5′-GGTCTTCTTTGTGTGCTTGGCACCCTACTATGTG-3′ (SEQ. ID. NO.:376) and theantisense primer had the following sequence:5′-CACATAGTAGGGTGCCAAGCACACAAAGAAGACC-3′ (SEQ. ID. NO.:377). Theendogenous GPR24 cDNA was used as a template.

I. F265I Mutation

Preparation of a non-endogenous version of the human GPR24 receptor wasaccomplished by creating an F265 I mutation (see, SEQ. ID. NO.:382 fornucleic acid sequence, and SEQ. ID. NO.:383 for amino acid sequence).Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit(Stratagene) according to the manufacturer. The PCR sense mutagenesisprimer used had the following sequence:5′-GCCATCTGTCTGGTCATCTTTGTGTGCTGGG-3′ (SEQ. ID. NO.:380) and theantisense primer had the following sequence:5′-CCCAGCACACAAAGATGACCAGACAGATGGC-3′ (SEQ. ID. NO.:381). The endogenousGPR24 cDNA was used as a template.

J. I261Q Mutation

Preparation of a non-endogenous version of the human GPR24 receptor wasaccomplished by creating an I 261Q mutation (see, SEQ. ID. NO.:386 fornucleic acid sequence, and SEQ. ID. NO.:387 for amino acid sequence).Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit(Stratagene) according to the manufacturer. The PCR sense mutagenesisprimer used had the following sequence:5′-CGCACAGCCATCGCCCAGTGTCTGGTCTTCTTTGTG-3′ (SEQ. ID. NO.:384) and theantisense primer had the following sequence:5′-CACAAAGAAGACCAGACACTGGGCGATGGCTGTGCG-3′ (SEQ. ID. NO.:385). Theendogenous GPR24 cDNA was used as a template.

K. D140N Mutation

Preparation of a non-endogenous version of the human GPR24 receptor wasaccomplished by creating an D140N mutation (see, SEQ. ID. NO.:390 fornucleic acid sequence, and SEQ. ID. NO.:391 for amino acid sequence).Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit(Stratagene) according to the manufacturer. The PCR sense mutagenesisprimer used had the following sequence:5′-ACCGCCATGGCCATTAACGCGTACCTGGCCACT-3′ (SEQ. ID. NO.:388) and theantisense primer had the following sequence:5′-AGTGGCCAGGTAGCGGTTAATGGCCATGGCGGT-3′ (SEQ. ID. NO.:389). Theendogenous GPR24 cDNA was used as a template.

3. TSHR

Preparation of non-endogenous versions of the human TSHR receptor wereaccomplished by creating an V509A mutation (see, SEQ. ID. NO.:394 fornucleic acid sequence, SEQ. ID. NO.:395 for amino acid sequence), aD619G mutation (see, SEQ. ID. NO.:398 for nucleic acid sequence, SEQ.ID. NO.:399 for amino acid sequence), an A623I mutation (see, SEQ. ID.NO.:402 for nucleic acid sequence, SEQ. ID. NO.:403 for amino acidsequence), a A623K mutation (see, SEQ. ID. NO.:406 for nucleic acidsequence, SEQ. ID. NO.:407 for amino acid sequence), an C672Y mutation(see, SEQ. ID. NO.:410 for nucleic acid sequence, SEQ. ID. NO.:411 foramino acid sequence), a D619G/A623K mutation (see, SEQ. ID. NO.:414 fornucleic acid sequence, SEQ. ID. NO.:415 for amino acid sequence), anV509A/C672Y mutation see, SEQ. ID. NO.:418 for nucleic acid sequence,SEQ. ID. NO.:419 for amino acid sequence), and an V509A/A623K/C672Ymutation (see, SEQ. ID. NO.:422 for nucleic acid sequence, SEQ. ID.NO.:423 for amino acid sequence).

A. V509A Mutation

Preparation of a non-endogenous version of the human TSHR receptor wasaccomplished by creating an V509A mutation (see, SEQ. ID. NO.:394 fornucleic acid sequence, and SEQ. ID. NO.:395 for amino acid sequence).Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit(Stratagene) according to the manufacturer. The PCR sense mutagenesisprimer used had the following sequence:5′-CAAGCGAGTTATCGGCATATACGCTGACGGTC-3′ (SEQ. ID. NO.:392) and theantisense primer had the following sequence:5′-GACCGTCAGCGTATATGCCGATAACTCGCTTG-3′ (SEQ. ID. NO.:393). Theendogenous TSHR cDNA was used as a template. This V509A mutant can bedifferentiated from the endogenous version by the absence of an AccIsite near the mutation site.

B. D619G Mutation

Preparation of a non-endogenous version of the human TSHR receptor wasalso accomplished by creating a D619G mutation (see, SEQ. ID. NO.:398for nucleic acid sequence, and SEQ. ID. NO.:399 for amino acidsequence). Mutagenesis was performed using QuikChange Site-DirectedMutagenesis Kit (Stratagene) according to the manufacturer. The PCRsense mutagenesis primer used had the following sequence:5′-ACCCAGGGGACAAAGGTACCAAAATTGCCAA-3′ (SEQ. ID. NO.:396) and theantisense primer had the following sequence:5′-TTGGCAATTTTGGTACCTTTGTCCCCTGGGT-3′ (SEQ. ID. NO.:397). The endogenousTSHR cDNA was used as a template. This D619G mutant can bedifferentiated from the endogenous version by the presence of a KpnIsite near the mutation site.

C. A623I Mutation

Preparation of a non-endogenous version of the human TSBR wasaccomplished by creating an A623I mutation (see, SEQ. ID. NO.:402 fornucleic acid sequence, and SEQ. ID. NO.:403 for amino acid sequence).Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit(Stratagene) according to the manufacturer. The PCR sense mutagenesisprimer used had the following sequence:5′-AAAGATACCAAAATTATCAAGAGGATGGCTGT-3′ (SEQ. ID. NO.:400) and theantisense primer had the following sequence:5′-ACAGCCATCCTCTTGATAATTTTGGTATCTTT-3′ (SEQ. ID. NO.:401). Theendogenous TSHR cDNA was used as a template. This A623I mutant can bedifferentiated from the endogenous version by the absence of a BstXIsite near the mutation site.

D. A623K Mutation

Preparation of a non-endogenous version of the human TSHR receptor wasalso accomplished by creating a A623K mutation (see, SEQ. ID. NO.:406for nucleic acid sequence, and SEQ. ID. NO.:407 for amino acidsequence). Mutagenesis was performed using QuikChange Site-DirectedMutagenesis Kit (Stratagene) according to the manufacturer. The PCRsense mutagenesis primer used had the following sequence:5′-AAAGATACCAAAATTAAGAAGAGGATGGCTGTG-3′ (SEQ. ID. NO.:404) and theantisense primer had the following sequence:5′-CACAGCCATCCTCTTCTTAATTTTGGTATCTTT-3′ (SEQ. ID. NO.:405). Theendogenous TSHR cDNA was used as a template. This A623K mutant can bedifferentiated from the endogenous version by the absence of a BstXIsite near the mutation site.

E. C672Y Mutation

Preparation of a non-endogenous version of the human TSHR receptor wasalso accomplished by creating a C672Y mutation (see, SEQ. ID. NO.:410for nucleic acid sequence, and SEQ. ID. NO.:411 for amino acidsequence). Mutagenesis was performed using QuikChange Site-DirectedMutagenesis Kit (Stratagene) according to the manufacturer. The PCRsense mutagenesis primer used had the following sequence:5′-CTATCCACTTAACTCGTACGCCAATCCATTCCTC-3′ (SEQ. ID. NO.:408) and theantisense primer had the following sequence:5′-GAGGAATGGATTGGCGTACGAGTTAAGTGGATAG-3′ (SEQ. ID. NO.:409). Theendogenous TSIR cDNA was used as a template. This C672Y mutant can bedifferentiated from the endogenous version by the presence of a BsiWIsite near the mutation site.

F. D619G/A623K Mutation

Preparation of a non-endogenous version of the human TSHR receptor wasalso accomplished by creating a D619G/A623K mutation (see, SEQ. ID.NO.:414 for nucleic acid sequence, and SEQ. ID. NO.:415 for amino acidsequence). Mutagenesis was performed using QuikChange Site-DirectedMutagenesis Kit (Stratagene) according to the manufacturer. The PCRsense mutagenesis primer used had the following sequence:

(SEQ.ID.NO.:412) 5′-ACCCAGGGGACAAAGGTACCAAAATTAAGAAGAGGATGGCTGTG-3′

and the antisense primer had the following sequence:5′-CACAGCCATCCTCTTCTTAATTTTGGTACCTTTGTCCCCTGGGT-3′ (SEQ. ID. NO.:413).The non-endogenous D619G mutant version of TSHR cDNA was used as atemplate. This D619G/A623K mutant can be differentiated from theendogenous version by the presence of a KpnI site near the D619Gmutation site and absence of a BstXI site near the A623K mutation site.

G. V509A/C672Y Mutation

Preparation of a non-endogenous version of the human TSHR receptor wasalso accomplished by creating a V509A/C672Y mutation (see, SEQ. ID.NO.:418 for nucleic acid sequence, and SEQ. ID. NO.:419 for amino acidsequence). Mutagenesis was performed using QuikChange Site-DirectedMutagenesis Kit (Stratagene) according to the manufacturer. The V509Asense mutagenesis primer used had the following sequence:5′-CAAGCGAGTTATCGGCATATACGCTGACGGTC-3′ (SEQ. ID. NO.:416) and the C672Yantisense primer had the following sequence:5′-GAGGAATGGATTGGCGTACGAGTTAAGTGGATAG-3′ (SEQ. ID. NO.:417). Theendogenous TSHR cDNA was used as a template. This V509A/C672Y mutant canbe differentiated from the endogenous version by the absence of an AccIsite near the V509A mutation site and presence of a BsiWI site near theC672Y mutation site.

H. V509A/A623K/C672Y Mutation

Preparation of a non-endogenous version of the human TSHR receptor wasalso accomplished by creating a V509A/A623K/C672Y mutation (see, SEQ.ID. NO.:422 for nucleic acid sequence, and SEQ. ID. NO.:423 for aminoacid sequence). Mutagenesis was performed using QuikChange Site-DirectedMutagenesis Kit (Stratagene) according to the manufacturer. The A623Ksense mutagenesis primer used had the following sequence:5′-AAAGATACCAAAATTAAGAAGAGGATGGCTGTG-3′ (SEQ. ID. NO.:420) and the A623Kantisense primer had the following sequence:5′-CACAGCCATCCTCTTCTTAATTTTGGTATCTTT-3′ (SEQ. ID. NO.:421). Thenon-endogenous V509A/C672Y mutant version of TSHR cDNA was used as atemplate. This V509A/A623K/C672Y mutant can be differentiated from theendogenous version by the absence of an AccI site near the V509Amutation site, absence of a BstXI near the A623K mutation site and thepresence of a BsiWI site near the C672Y mutation 10 site.

The non-endogenous human GPCRs were then sequenced and the derived andverified nucleic acid and amino acid sequences are listed in theaccompanying “Sequence Listing” appendix to this patent document, assummarized in Table G below:

TABLE G Nucleic Acid Amino Acid Mutated GPCR Sequence Listing SequenceListing 5HT-1A SEQ.ID.NO.:424 SEQ.ID.NO.:425 V343K 5HT-1B SEQ.ID.NO.:426SEQ.ID.NO.:427 T313K 5HT-1D SEQ.ID.NO.:428 SEQ.ID.NO.:429 T300K 5HT-1ESEQ.ID.NO.:430 SEQ.ID.NO.:431 A290K 5HT-1F SEQ.ID.NO.:432 SEQ.ID.NO.:433A292K 5HT-2B SEQ.ID.NO.:434 SEQ.ID.NO.:435 S323K 5HT-4A SEQ.ID.NO.:436SEQ.ID.NO.:437 A258K 5HT-4B SEQ.ID.NO.:438 SEQ.ID.NO.:439 A258K 5HT-4CSEQ.ID.NO.:440 SEQ.ID.NO.:441 A258K 5HT-4D SEQ.ID.NO.:442 SEQ.ID.NO.:443A258K 5HT-4E SEQ.ID.NO.:444 SEQ.ID.NO.:445 A258K 5HT-5A SEQ.ID.NO.:446SEQ.ID.NO.:447 A284K 5HT-6 SEQ.ID.NO.:448 SEQ.ID.NO.:449 S267K 5HT-7SEQ.ID.NO.:450 SEQ.ID.NO.:451 A326K AVPR1A SEQ.ID.NO.:452 SEQ.ID.NO.:453V290K AVPR1B SEQ.ID.NO.:454 SEQ.ID.NO.:455 V280K AVPR2 SEQ.ID.NO.:456SEQ.ID.NO.:457 V270K BBR3 SEQ.ID.NO.:458 SEQ.ID.NO.:459 A270K BDKR1SEQ.ID.NO.:460 SEQ.ID.NO.:461 T249K BDKR2 SEQ.ID.NO.:462 SEQ.ID.NO.:463T269K C3a SEQ.ID.NO.:464 SEQ.ID.NO.:465 F376K C5a SEQ.ID.NO.:466SEQ.ID.NO.:467 L241K CB1 SEQ.ID.NO.:468 SEQ.ID.NO.:469 A342K CB2SEQ.ID.NO.:470 SEQ.ID.NO.:471 A244K CCR2b SEQ.ID.NO.:472 SEQ.ID.NO.:473V242K CCR3 SEQ.ID.NO.:474 SEQ.ID.NO.:475 I238K CCR5 SEQ.ID.NO.:476SEQ.ID.NO.:477 V234K CCR8 SEQ.ID.NO.:478 SEQ.ID.NO.:479 I237K CCR9SEQ.ID.NO.:480 SEQ.ID.NO.:481 L253K CRFR1 SEQ.ID.NO.:482 SEQ.ID.NO.:483T316P CXCR4 SEQ.ID.NO.:484 SEQ.ID.NO.:485 L238K Dopamine D1SEQ.ID.NO.:486 SEQ.ID.NO.:487 L271K Dopamine D2 SEQ.ID.NO.:488SEQ.ID.NO.:489 T372K Dopamine D3 SEQ.ID.NO.:490 SEQ.ID.NO.:491 T328KDopamine D5 SEQ.ID.NO.:492 SEQ.ID.NO.:493 L295K ETA SEQ.ID.NO.:494SEQ.ID.NO.:495 A305K ETB SEQ.ID.NO.:496 SEQ.ID.NO.:497 A322K FPR1SEQ.ID.NO.:498 SEQ.ID.NO.:499 L240K FPRL1 SEQ.ID.NO.:500 SEQ.ID.NO.:501L240K GALR1 SEQ.ID.NO.:502 SEQ.ID.NO.:503 A246K GALR2 SEQ.ID.NO.:504SEQ.ID.NO.:505 T235K GIP SEQ.ID.NO.:506 SEQ.ID.NO.:507 T343P mGluR1SEQ.ID.NO.:346 SEQ.ID.NO.:347 3′ Deletion GPR5 SEQ.ID.NO.:508SEQ.ID.NO.:509 V224K GPR24 (also known as MCH or SLC-1) T255KSEQ.ID.NO.:350 SEQ.ID.NO.:351 T255K/T257R SEQ.ID.NO.:354 SEQ.ID.NO.:35524-IC3-SST2 SEQ.ID.NO.:358 SEQ.ID.NO.:359 C305Y SEQ.ID.NO.:362SEQ.ID.NO.:363 P271L SEQ.ID.NO.:366 SEQ.ID.NO.:367 W269C SEQ.ID.NO.:370SEQ.ID.NO.:371 W269F SEQ.ID.NO.:374 SEQ.ID.NO.:375 W269L SEQ.ID.NO.:378SEQ.ID.NO.:379 F265I SEQ.ID.NO.:382 SEQ.ID.NO.:383 I261Q SEQ.ID.NO.:386SEQ.ID.NO.:387 D140N SEQ.ID.NO.:390 SEQ.ID.NO.:391 GRPR SEQ.ID.NO.:510SEQ.ID.NO.:511 A263K M1 SEQ.ID.NO.:512 SEQ.ID.NO.:513 A364K M2SEQ.ID.NO.:514 SEQ.ID.NO.:515 T386K M3 SEQ.ID.NO.:516 SEQ.ID.NO.:517A490K M4 SEQ.ID.NO.:518 SEQ.ID.NO.:519 T399K M5 SEQ.ID.NO.:520SEQ.ID.NO:521 A441K MC3 SEQ.ID.NO.:522 SEQ.ID.NO.:523 A241K NK1RSEQ.ID.NO.:524 SEQ.ID.NO.:525 V247K NK2R SEQ.ID.NO.:526 SEQ.ID.NO.:527V249K NK3R SEQ.ID.NO.:528 SEQ.ID.NO.:529 V298K NMBR SEQ.ID.NO.:530SEQ.ID.NO.:531 A265K NPY5 SEQ.ID.NO.:532 SEQ.ID.NO.:533 A297K NTSR1SEQ.ID.NO.:534 SEQ.ID.NO.:535 V302K NTSR2 SEQ.ID.NO.:536 SEQ.ID.NO.:537V269K OPRD SEQ.ID.NO.:538 SEQ.ID.NO.:539 T260K OPRL1 SEQ.ID.NO.:540SEQ.ID.NO.:541 T262K OPRK SEQ.ID.NO.:542 SEQ.ID.NO.:543 T273K OPRMSEQ.ID.NO.:544 SEQ.ID.NO.:545 T281K OPRM1A SEQ.ID.NO.:546 SEQ.ID.NO.:547T281K OX₁R SEQ.ID.NO.:548 SEQ.ID.NO.:549 F367K OX₂R SEQ.ID.NO.:550SEQ.ID.NO.:551 A297K PACAP SEQ.ID.NO.:552 SEQ.ID.NO.:553 T355K PAFSEQ.ID.NO.:554 SEQ.ID.NO.:555 L231K PGE EP1 SEQ.ID.NO.:556SEQ.ID.NO.:557 V296K PGE EP2 SEQ.ID.NO.:558 SEQ.ID.NO.:559 L263K PGE EP4SEQ.ID.NO.:560 SEQ.ID.NO.:561 V271K PTHR1 SEQ.ID.NO.:562 SEQ.ID.NO.:563T410P PTHR2 SEQ.ID.NO.:564 SEQ.ID.NO.:565 T365P SCTR SEQ ID.NO.:566SEQ.ID.NO.:567 T344P SST1 SEQ.ID.NO.:568 SEQ.ID.NO.:569 T290K SST2SEQ.ID.NO.:570 SEQ.ID.NO.:571 T255K SST3 SEQ.ID.NO.:572 SEQ.ID.NO.:573T256K SST4 SEQ.ID.NO.:574 SEQ.ID.NO.:575 T258K SST5 SEQ.ID.NO.:576SEQ.ID.NO.:577 T247K TSHR V509A SEQ.ID.NO.:394 SEQ.ID.NO.:395 D619GSEQ.ID.NO.:398 SEQ.ID.NO.:399 A623I SEQ.ID.NO.:402 SEQ.ID.NO.:403 A623KSEQ.ID.NO.:406 SEQ.ID.NO.:407 C672Y SEQ.ID.NO.:410 SEQ.ID.NO.:411D619G/A623K SEQ.ID.NO.:414 SEQ.ID.NO.:415 V509A/C672Y SEQ.ID.NO.:418SEQ.ID.NO.:419 V509A/A623K1C672Y SEQ.ID.NO.:422 SEQ ID.NO.:423 VIPRSEQ.ID.NO.:578 SEQ.ID.NO.:579 T343P VIPR2 SEQ.ID.NO.:580 SEQ.ID.NO.:581T330P

Assessment of constitutive activity of the non-endogenous versions ofthe known GPCRs can then be accomplished.

Example 3

Receptor Expression

Although a variety of cells are available to the art for the expressionof proteins, it is most preferred that mammalian cells be utilized. Theprimary reason for this is predicated upon practicalities, i.e.,utilization of, e.g., yeast cells for the expression of a GPCR, whilepossible, introduces into the protocol a non-mammalian cell which maynot (indeed, in the case of yeast, does not) include thereceptor-coupling, genetic-mechanism and secretary pathways that haveevolved for mammalian systems—thus, results obtained in non-mammaliancells, while of potential use, are not as preferred as that obtainedfrom mammalian cells. Of the mammalian cells, COS-7, Hek-293 andHek-293T cells are particularly preferred, although the specificmammalian cell utilized can be predicated upon the particular needs ofthe artisan. The following approach was used for the indicatedreceptors, and can also be applied with respect to other receptorsdisclosed herein.

On day one, 2×10⁴ Hek-293T cells well were plated out. On day two, tworeaction tubes were prepared (the proportions to follow for each tubeare per plate): tube A was prepared by mixing 20 μg DNA (e.g., pCMVvector; pCMV vector with receptor cDNA, etc.) in 1.2 ml serum free DMEM(Irvine Scientific, Irvine, Calif.); tube B was prepared by mixing 120μl lipofectamine (Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and Bwere admixed by inversions (several times), followed by incubation atroom temperature for 30-45 min. The admixture is referred to as the“transfection mixture”. Plated Hek-293T cells were washed with 1×PBS,followed by addition of 10 ml serum free DMEM. 2.4 ml of thetransfection mixture were added to the cells, followed by incubation for4 hrs at 37° C./5% CO₂. The transfection mixture was removed byaspiration, followed by the addition of 25 ml of DMEM/10% Fetal BovineSerum. Cells were incubated at 37° C./5% CO₂. After 72 hr incubation,cells were harvested and utilized for analysis.

Example 4

Assays for Determination of Constitutive Activity of Non-EndogenousGPCRS

A variety of approaches are available for assessment of constitutiveactivity of the non-endogenous versions of known GPCRs. The followingare illustrative; those of ordinary skill in the art are credited withthe ability to determine those techniques that are preferentiallybeneficial for the needs of the artisan.

1. Membrane Binding Assays: [³⁵S]GTPγS Assay

When a G protein-coupled receptor is in its active state, either as aresult of ligand binding or constitutive activation, the receptorcouples to a G protein and stimulates the release of GDP and subsequentbinding of GTP to the G protein. The alpha subunit of the Gprotein-receptor complex acts as a GTPase and slowly hydrolyzes the GTPto GDP, at which point the receptor normally is deactivated.Constitutively activated receptors continue to exchange GDP for GTP. Thenon-hydrolyzable GTP analog, [³⁵5]GTPγS, can be utilized to demonstrateenhanced binding of [³⁵S]GTPγS to membranes expressing constitutivelyactivated receptors. The advantage of using [³⁵S]GTPγS binding tomeasure constitutive activation is that: (a) it is genericallyapplicable to all G protein-coupled receptors; (b) it is proximal at themembrane surface making it less likely to pick-up molecules which affectthe intracellular cascade.

The assay utilizes the ability of G protein coupled receptors tostimulate [³⁵S]GTPγS binding to membranes expressing the relevantreceptors. The assay can, therefore, be used in the directidentification method to screen candidate compounds to known, andconstitutively activated G protein-coupled receptors. The assay isgeneric and has application to drug discovery at all G protein-coupledreceptors.

The [³⁵S]GTPγS assay can be incubated in 20 mM HEPES and between 1 andabout 20 mM MgCl₂ (this amount can be adjusted for optimization ofresults, although 20 mM is preferred) pH 7.4, binding buffer withbetween about 0.3 and about 1.2 nM [³⁵S]GTPγS (this amount can beadjusted for optimization of results, although 1.2 is preferred) and12.5 to 75 μg membrane protein (e.g. COS-7 cells expressing thereceptor; this amount can be adjusted for optimization, although 75 μgis preferred) and 1 μM GDP (this amount can be changed for optimization)for 1 hour. Wheatgerm agglutinin beads (25 μl; Amersham) should then beadded and the mixture incubated for another 30 minutes at roomtemperature. The tubes are then centrifuged at 1500×g for 5 minutes atroom temperature and then counted in a scintillation counter.

A less costly but equally applicable alternative has been identifiedwhich also meets the needs of large scale screening. FLASH PLATES™ andWALLAC™ scintistrips may be utilized to format a high throughput[³⁵S]GTPγS binding assay. Furthermore, using this technique, the assaycan be utilized for known GPCRs to simultaneously monitor tritiatedligand binding to the receptor at the same time as monitoring theefficacy via [³⁵S]GTPγS binding. This is possible because the Wallacbeta counter can switch energy windows to look at both tritium and³⁵S-labeled probes. This assay may also be used to detect other types ofmembrane activation events resulting in receptor activation. Forexample, the assay may be used to monitor ³²P phosphorylation of avariety of receptors (both G protein coupled and tyrosine kinasereceptors). When the membranes are centrifuged to the bottom of thewell, the bound [³⁵S]GTPγS or the ³²P-phosphorylated receptor willactivate the scintillant which is coated of the wells. Scinti® strips(Wallac) have been used to demonstrate this principle. In addition, theassay also has utility for measuring ligand binding to receptors usingradioactively labeled ligands. In a similar manner, when theradiolabeled bound ligand is centrifuged to the bottom of the well, thescintistrip label comes into proximity with the radiolabeled ligandresulting in activation and detection.

2. Membrane-Based cAMP

A FLASH PLATE™ Adenylyl Cyclase kit (New England Nuclear; Cat. No.SMP004A) designed for cell-based assays can be modified for use withcrude plasma membranes. The Flash Plate wells contain a scintillantcoating which also contains a specific antibody recognizing cAMP. ThecAMP generated in the wells was quantitated by a direct competition forbinding of radioactive cAMP tracer to the cAMP antibody. The followingserves as a brief protocol for the measurement of changes in cAMP levelsin membranes that express the receptors.

Transfected cells are harvested approximately three days aftertransfection. Membranes were prepared by homogenization of suspendedcells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl₂.Homogenization is performed on ice using a Brinkman POLYTRON™ forapproximately 10 seconds. The resulting homogenate is centrifuged at49,000×g for 15 minutes at 4° C. The resulting pellet is thenresuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA,homogenized for 10 seconds, followed by centrifugation at 49,000×g for15 minutes at 4° C. The resulting pellet can be stored at −80° C. untilutilized. On the day of measurement, the membrane pellet is slowlythawed at room temperature, resuspended in buffer containing 20 mMHEPES, pH 7.4 and 10 mM MgCl₂ (these amounts can be optimized, althoughthe values listed herein are preferred), to yield a final proteinconcentration of 0.60 mg/ml (the resuspended membranes were placed onice until use).

cAMP standards and Detection Buffer (comprising 2 μCi of tracer [¹²⁵IcAMP (100 μl)] to 11 ml Detection Buffer) are prepared and maintained inaccordance with the manufacturer's instructions. Assay Buffer isprepared fresh for screening and contained 20 mM HEPES, pH 7.4, 10 mMMgCl₂, 20 mM (Sigma), 0.1 units/ml creatine phosphokinase (Sigma), 50 μMGTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer can be stored on iceuntil utilized. The assay is initiated by the addition of 50 μL of assaybuffer followed by addition of 50 μL of membrane suspension to the NENFlash Plate. The resultant assay mixture is incubated for 60 minutes atroom temperature followed by addition of 100 μL of detection buffer.Plates are then incubated an additional 2-4 hours followed by countingin a Wallac MicroBeta™ scintillation counter. Values of cAMP/well areextrapolated from a standard cAMP curve that is contained within eachassay plate.

3. Cell-Based cAMP for Gi Coupled Target GPCRs

TSHR is a Gs coupled GPCR that causes the accumulation of cAMP uponactivation. TSHR was constitutively activated by mutating amino acidresidue 623 (i.e., changing an alanine residue to an isoleucineresidue). See, SEQ. ID. NO.:402 for nucleic acid sequence and SEQ. ID.NO.:403 for deduced amino acid sequence. A Gi coupled receptor isexpected to inhibit adenylyl cyclase, and, therefore, decrease the levelof cAMP production, which can make assessment of cAMP levelschallenging. An effective technique for measuring the decrease inproduction of cAMP as an indication of constitutive activation of a Gicoupled receptor can be accomplished by co-transfecting, mostpreferably, non-endogenous, constitutively activated TSHR (TSHR-A623I)(or an endogenous, constitutively active Gs coupled receptor) as a“signal enhancer” with a Gi linked target GPCR, such as GPR24, toestablish a baseline level of cAMP. Upon creating a non-endogenousversion of the Gi coupled receptor, this non-endogenous version of thetarget GPCR is then co-transfected with the signal enhancer, and it isthis material that can be used for screening. We utilized such approachto effectively generate a signal when a cAMP assay is used; thisapproach is preferably used in the direct identification of candidatecompounds against Gi coupled receptors. It is noted that for a Gicoupled GPCR, when this approach is used, an inverse agonist of thetarget GPCR will increase the cAMP signal and an agonist will decreasethe cAMP signal.

On day one, 2×10⁴Hek-293 and Hek-293T cells/well were plated out. On daytwo, two reaction tubes were prepared (the proportions to follow foreach tube are per plate): tube A was prepared by mixing 2 μg DNA of eachreceptor transfected into the mammalian cells, for a total of 4 μg DNA(e.g., pCMV vector; pCMV vector with mutated THSR (TSHR-A6231);TSHR-A623I and GPR24, etc.) in 1.2 ml serum free DMEM (IrvineScientific, Irvine, Calif.); tube B was prepared by mixing 120 μllipofectamine (Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and B werethen admixed by inversion (several times), followed by incubation atroom temperature for 30-45 min. The admixture is referred to as the“transfection mixture”. Plated Hek-293 cells were washed with 1×PBS,followed by addition of 10 ml serum free DMEM. 2.4 ml of thetransfection mixture was then added to the cells, followed by incubationfor 4 hrs at 37° C./5% CO₂. The transfection mixture was then removed byaspiration, followed by the addition of 25 ml of DMEM/10% Fetal BovineSerum. Cells were then incubated at 37° C./5% CO₂. After 24 hrincubation, cells were then harvested and utilized for analysis.

A FLASH PLATE™ Adenylyl Cyclase kit (New England Nuclear; Cat. No.SMP004A) designed for cell-based assays can be modified for use withcrude plasma membranes. The Flash Plate wells can contain a scintillantcoating which also contains a specific antibody recognizing cAMP. ThecAMP generated in the wells can be quantitated by a direct competitionfor binding of radioactive cAMP tracer to the cAMP antibody. Thefollowing serves as a brief protocol for the measurement of changes incAMP levels in whole cells that express the receptors.

Transfected cells were harvested approximately twenty four hours aftertransient transfection. Media was carefully aspirated and discarded. Tenmilliliters of PBS was gently added to each dish of cells followed bycareful aspiration. One milliliter of Sigma cell dissociation buffer and3 ml of PBS are added to each plate. Cells were pipetted off the plateand the cell suspension is collected into a 50 ml conical centrifugetube. Cells were then centrifuged at room temperature at 1,100 rpm for 5min. The cell pellet was carefully re-suspended into an appropriatevolume of PBS (about 3 ml/plate). The cells were then counted using ahemocytometer and additional PBS is added to give the appropriate numberof cells (with a final concentration of about 50 μl/well).

cAMP standards and Detection Buffer (comprising 1 μCi of tracer [¹²⁵IcAMP (50 μl] to 11 ml Detection Buffer) was prepared and maintained inaccordance with the manufacturer's instructions. Assay Buffer should beprepared fresh for screening and contained 50 μL of Stimulation Buffer,3 μL of test compound (12 μM final assay concentration) and 50 μL cells,Assay Buffer can be stored on ice until utilized. The assay can beinitiated by addition of 50 μL of cAMP standards to appropriate wellsfollowed by addition of 50 μL of PBSA to wells H-11 and H12. Fifty μL ofStimulation Buffer was added to all wells. Selected compounds (e.g.,TSH, 100 nM MCH, MCH/TSH) were added to appropriate wells using a pintool capable of dispensing 3 μL of compound solution, with a final assayconcentration of 12 μM test compound and 100 μL total assay volume. Thecells were then added to the wells and incubated for 60 min at roomtemperature. 100 μL of Detection Mix containing tracer cAMP was thenadded to the wells. Plates were then incubated additional 2 hoursfollowed by counting in a Wallac MicroBeta scintillation counter. Valuesof cAMP/well were then extrapolated from a standard cAMP curve which iscontained within each assay plate.

FIG. 1 evidences about a 22% decrease in cAMP production of cellsco-transfected with TSHR-A623I (in the presence of TSH) andnon-endogenous, constitutively activated GPR24 (“24-IC3-SST2”) (262.266pmol cAMP/well) compared to TSHR-A623I with endogenous GPR24 (“GPR24wt”) (336.50293 pmol cAMP/well). Co-transfection of TSHR-A623I withnon-endogenous, constitutively activated GPR24 (“I261Q”) evidences abouta 27% decrease in production of cAMP when compared to “GPR24 wt.” Such adecrease in cAMP production signifies that non-endogenous version ofGPR24 (“I261Q”) is constitutively active. Thus, a candidate compoundwhich impacts the GPR24 receptor by increasing the cAMP signal is aninverse agonist, while a GPR24 agonist will decrease the cAMP signal.Based upon the data generated for FIG. 1, 24-IC3-SST2 and I261Q are mostpreferred non-endogenous versions of GPR24 when used in a TSHR(constitutively activated co-transfection approach using a cAMP assay.

FIG. 2 evidences about a 60% decrease in cAMP production of cellsco-transfected with TSHR-A623I (in the presence of TSH) andnon-endogenous, constitutively activated GPR5 (“V224K”) (23.5 pmolecAMP/well) compared to TSHR-A623I with endogenous GPR5 (“GPR5 wt”)(58.79 pmol cAMP/well). About a 78% and about a 45% decrease inproduction of cAMP was evidenced when comparing TSHR-A6231co-transfected with “V225K” and TSHR-A623I co-transfected with “GPR5 wt”against pCMV co-transfected with TSHR-A623I (106.75 pmol cAMP/well),respectively. As mentioned above, a decrease in cAMP productionevidences a constitutively active GPR5. Thus, a preferred candidatecompound (i.e., an inverse agonist) would likely bind the Gi coupledreceptor to increase the signal of activation.

Preferably, and as noted previously, to ensure that a small moleculecandidate compound is targeting the Gi coupled target receptor and not,for example, the TSHR-A623I, the directly identified candidate compoundis preferably screened against the signal enhancer in the absence of thetarget receptor.

C. Reporter-Based Assays

1. CRE-Luc Reporter Assay (Gs-Associated Receptors)

A method to detect Gs stimulation depends on the known property of thetranscription factor CREB, which is activated in a cAMP-dependentmanner. A PATHDETECT™ CREB trans-Reporting System (Stratagene, Catalogue#219010) can utilized to assay for Gs coupled activity in 293 or 293Tcells. Cells are transfected with the plasmids components of this abovesystem and the indicated expression plasmid encoding endogenous ormutant receptor using a Mammalian Transfection Kit (Stratagene,Catalogue #200285) according to the manufacturer's instructions.Briefly, 400 ng pFR-Luc (luciferase reporter plasmid containing GaL4recognition sequences), 40 ng pFA2-CREB (Ga14-CREB fusion proteincontaining the Ga14 DNA-binding domain), 80 ng pCMV-receptor expressionplasmid (comprising the receptor) and 20 ng CMV-SEAP (secreted alkalinephosphatase expression plasmid; alkaline phosphatase activity ismeasured in the media of transfected cells to control for variations intransfection efficiency between samples) are combined in a calciumphosphate precipitate per the Kit's instructions. Half of theprecipitate is equally distributed over 3 wells in a 96-well plate, kepton the cells overnight, and replaced with fresh medium the followingmorning. Forty-eight (48) hr after the start of the transfection, cellsare treated and assayed for, e.g., luciferase activity.

2. 8XCRE-Luc Reporter Assay

HEK-293T cells are plated-out on 96 well plates at a density of 3×10⁴cells per well and were transfected using Lipofectamine Reagent (BRL)the following day according to manufacturer instructions. A DNA/lipidmixture is prepared for each 6-well transfection as follows: 260 ng ofplasmid DNA in 100 μl of DMEM were gently mixed with 2 μl of lipid in100 μl of DMEM (the 260 ng of plasmid DNA consisted of 200 ng of a8xCRE-Luc reporter plasmid (see below and FIG. 1 for a representation ofa portion of the plasmid), 50 ng of pCMV comprising endogenous receptoror non-endogenous receptor or pCMV alone, and long of a GPRS expressionplasmid (GPRS in pcDNA3 (Invitrogen)). The 8XCRE-Luc reporter plasmidwas prepared as follows: vector SRIF-β-gal was obtained by cloning therat somatostatin promoter (−71/+51) at BglV-HindIII site in thepβgal-Basic Vector (Clontech). Eight (8) copies of cAMP response elementwere obtained by PCR from an adenovirus template AdpCF126CCRE8 (see, 7Human Gene Therapy 1883 (1996)) and cloned into the SRIF-β-gal vector atthe Kpn-BglV site, resulting in the 8xCRE-β-gal reporter vector. The8xCRE-Luc reporter plasmid was generated by replacing thebeta-galactosidase gene in the 8xCRE-p-gal reporter vector with theluciferase gene obtained from the pGL3-basic vector (Promega) at theHindIII-BamHI site. Following 30 min. incubation at room temperature,the DNA/lipid mixture was diluted with 400 μl of DMEM and 100 μl of thediluted mixture was added to each well. 100 μl of DMEM with 10% FCS wereadded to each well after a 4 hr incubation in a cell culture incubator.The following day the transfected cells were changed with 200 μl/well ofDMEM with 10% FCS. Eight (8) hours later, the wells were changed to 100μl/well of DMEM without phenol red, after one wash with PBS. Luciferaseactivity were measured the next day using the LucLite™ reporter geneassay kit (Packard) following manufacturer instructions and read on a1450 MicroBeta™ scintillation and luminescence counter (Wallac).

FIG. 3A represents about a 63% increase in activity of thenon-endogenous, constitutively active version of human Dopamine D1receptor (189270 relative light units) compared with that of theendogenous Dopamine D1 (70622 relative light units).

FIG. 3B represents about a 48% decreases in activity of thenon-endogenous, constitutively active version of human OPRM (a Gicoupled receptor; see Example 4(3)), about a 53% decrease in activity ofthe non-endogenous, constitutively active version of human 5-HT1A (a Gicoupled receptor; see Example 4(3)), about a 91% increase in activity ofthe non-endogenous, constitutively active version of human 5-HT1B, andabout a 20% increase in activity of the non-endogenous, constitutivelyactive version of human 5-HT2B over the respective endogenous version ofthe GPCR.

FIG. 3C represents about a 29% increase in activity of thenon-endogenous, constitutively active version of human CCR3, about a 41%increase in activity of the non-endogenous, constitutively activeversion of human NTSR1, about a 51% increase in activity of thenon-endogenous, constitutively active version of human CB2, and about a40% decrease in activity of the non-endogenous, constitutively activeversion of human CXCR4 (a Gi coupled receptor; see Example 4(3)) overthe respective endogenous version of the GPCR.

FIG. 3D represents about a 75% increase in activity of thenon-endogenous, constitutively active version of human PTHR1, about a74% increase in activity of the non-endogenous, constitutively activeversion of human PTHR2, about a 56% increase in activity of thenon-endogenous, constitutively active version of human SCTR, about a 96%increase in activity of the non-endogenous, constitutively activeversion of human PACAP, about a 88% increase in activity of thenon-endogenous, constitutively active version of human VIPR1, and abouta 91% increase in activity of the non-endogenous, constitutively activeversion of human VIPR2 over the respective endogenous version of theGPCR.

FIG. 3E represents about a 51% increase in activity of thenon-endogenous, constitutively active version of human NTSR1, about a31% decrease in activity of the non-endogenous, constitutively activeversion of human M1, about a 19% decrease in activity of thenon-endogenous, constitutively active version of human M2, about a 32%increase in activity of the non-endogenous, constitutively activeversion of human M3, about a 33% decrease in activity of thenon-endogenous, constitutively active version of human M4, about a 17%decrease in activity of the non-endogenous, constitutively activeversion of human M5, and about a 60% increase in activity of thenon-endogenous, constitutively active version of human 5-HT1D over therespective endogenous version of the GPCR. M2, M4 and 5-HT1D areindicated as being Gi coupled while NTSR1, M1, M3 and M5 are indicatedas being Gq coupled.

3. AP1 Reporter Assay (Gq-Associated Receptors)

A method to detect Gq stimulation depends on the known property ofGq-dependent phospholipase C to cause the activation of genes containingAP1 elements in their promoter. A PATHDETECT™ AP-1 cis-Reporting System(Stratagene, Catalog #219073) can be utilized following the protocol setforth above with respect to the CREB reporter assay, except that thecomponents of the calcium phosphate precipitate were 410 ng pAP1-Luc, 80ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP.

4. SRF-Luc Reporter Assay (Gq-Associated Receptors)

One method to detect Gq stimulation depends on the known property ofGq-dependent phospholipase C to cause the activation of genes containingserum response factors in their promoter. A PATHDETECT™SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gqcoupled activity in, e.g., COS7 cells. Cells are transfected with theplasmid components of the system and the indicated expression plasmidencoding endogenous or non-endogenous GPCR using a MAMMALIANTRANSFECTION™ Kit (Stratagene, Catalogue #200285) according to themanufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ngpCMV-receptor expression plasmid and 20 ng CMV-SEAP (secreted alkalinephosphatase expression plasmid; alkaline phosphatase activity ismeasured in the media of transfected cells to control for variations intransfection efficiency between samples) are combined in a calciumphosphate precipitate as per the manufacturer's instructions. Half ofthe precipitate is equally distributed over 3 wells in a 96-well plate,kept on the cells in a serum free media for 24 hours. The last 5 hoursthe cells are incubated with 1 μM Angiotensin, where indicated. Cellsare then lysed and assayed for luciferase activity using a LUCLITE™ Kit(Packard, Cat. #6016911) and “Trilux 1450 Microbeta” liquidscintillation and luminescence counter (Wallac) per the manufacturer'sinstructions. The data can be analyzed using GraphPad Prism™ 2.0a(GraphPad Software Inc.).

5. Intracellular IP₃ Accumulation Assay (Gq-Associated Receptors)

On day 1, cells comprising the receptors (endogenous and/ornon-endogenous) can be plated onto 24 well plates, usually 1×10⁵cells/well (although this number can be optimized. On day 2 cells can betransfected by firstly mixing 0.25 μg DNA in 50 μL serum free DMEM/welland 2 μL lipofectamine in 50 μl serum-free DMEM/well. The solutions aregently mixed and incubated for 15-30 min at room temperature. Cells arewashed with 0.5 ml PBS and 400 μl of serum free media is mixed with thetransfection media and added to the cells. The cells are then incubatedfor 3-4 hrs at 37° C./5% CO₂ and then the transfection media is removedand replaced with 1 ml/well of regular growth media. On day 3 the cellsare labeled with ³H-myo-inositol. Briefly, the media is removed and thecells are washed with 0.5 ml PBS. Then 0.5 ml inositol-free/serum freemedia (GIBCO BRL) is added/well with 0.25 μCi of ³H-myo-inositol/welland the cells are incubated for 16-18 hrs o/n at 37° C./5% CO₂. On Day 4the cells are washed with 0.5 ml PBS and 0.45 ml of assay medium isadded containing inositol-free/serum free media 10 μM pargyline 10 mMlithium chloride or 0.4 ml of assay medium and 50 μL of 10× ketanserin(ket) to final concentration of 10 μM. The cells are then incubated for30 min at 37° C. The cells are then washed with 0.5 ml PBS and 200 μL offresh/ice cold stop solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) isadded/well. The solution is kept on ice for 5-10 min or until cells werelysed and then neutralized by 200 μl of fresh/ice cold neutralizationsol. (7.5% HCL). The lysate is then transferred into 1.5 ml eppendorftubes and 1 ml of chloroform/methanol (1:2) is added/tube. The solutionis vortexed for 15 sec and the upper phase is applied to a BioradAG1-X8™ anion exchange resin (100-200 mesh). Firstly, the resin iswashed with water at 1:1.25 W/V and 0.9 ml of upper phase is loaded ontothe column. The column is washed with 10 mls of 5 mM myo-inositol and 10ml of 5 mM Na-borate/60 mM Na-formate. The inositol tris phosphates areeluted into scintillation vials containing 10 ml of scintillationcocktail with 2 ml of 0.1 M formic acid/1 M ammonium formate. Thecolumns are regenerated by washing with 10 ml of 0.1 M formic acid/3Mammonium formate and rinsed twice with H₂O and stored at 4° C. in water.

FIG. 4 represents two preferred non-endogenous, constitutively activatedexemplary versions of GPR24, 24-IC3-SST2 and I261Q, for use in an IP3assay. When compared to the endogenous version of GPR24 (“GPR24 wt”),24-IC3-SST2 evidenced about a 27% increase in IP₃ accumulation, whilethe I26Q version represented and about a 32% increase.

Example 6

GPCR Fusion Protein Preparation

The design of the constitutively activated GPCR-G protein fusionconstruct was accomplished as follows: both the 5′ and 3′ ends of therat G protein Gsoa (long form; Itoh, H. et al., 83 PNAS 3776 (1986))were engineered to include a HindIII (5′-AAGCTT-3′) sequence thereon.Following confirmation of the correct sequence (including the flankingHindIII sequences), the entire sequence was shuttled into pcDNA3.1(−)(Invitrogen, cat. no. V795-20) by subcloning using the HindIIIrestriction site of that vector. The correct orientation for the Gsu.sequence was determined after subcloning into pcDNA3.1(−). The modifiedpcDNA3.1(−) containing the rat Gscc gene at HindIII sequence was thenverified; this vector was now available as a “universal” Gscc proteinvector. The pcDNA3.1 (−) vector contains a variety of well-knownrestriction sites upstream of the HindIII site, thus beneficiallyproviding the ability to insert, upstream of the Gs protein, the codingsequence of an endogenous, constitutively active GPCR. This sameapproach can be utilized to create other “universal” G protein vectors,and, of course, other commercially available or proprietary vectorsknown to the artisan can be utilized—the important criteria is that thesequence for the GPCR be upstream and in-frame with that of the Gprotein.

1. TSHR-Gsα Fusion Protein

a. Stable Cell Line Production for TSHR

Approximately 1.2 to 1.3×10⁷ HEK-293 cells are plated on a 15 cm tissueculture plate. Grown in DME High Glucose Medium containing ten percentfetal bovine serum and one percent sodium pyruvate, L-glutamine, andantibiotics. Twenty-four hours following plating of 293 cells to ˜80%confluency, the cells are transfected using 12 μg of DNA. The 12 μg ofDNA is combined with 60 μL of lipofectamine and 2 mL of DME High GlucoseMedium without serum. The medium is aspirated from the plates and thecells are washed once with medium without serum. The DNA, lipofectamine,and medium mixture is added to the plate along with 10 mL of mediumwithout serum. Following incubation at 37° C. for four to five hours,the medium is aspirated and 25 ml of medium containing serum is added.Twenty-four hours following transfection, the medium is aspirated again,and fresh medium with serum is added. Forty-eight hours followingtransfection, the medium is aspirated and medium with serum is addedcontaining geneticin (G418 drug) at a final concentration of 500 μg/mL.The transfected cells now undergo selection for positively transfectedcells containing the G418 resistant gene. The medium is replaced everyfour to five days as selection occurs. During selection, cells are grownto create stable pools, or split for stable clonal selection.

b. TSHR(A623K) Fusion Protein

TSHR-Gsα Fusion Protein construct was then made as follows: primers weredesigned for both endogenous, constitutively activated andnon-endogenous, constitutively activated TSAR were as follows:

(SEQ.ID.NO.:582; sense)     5′-gatc[TCTAGA]ATGAGGCCGGCGGACTTGCTGC-3′(SEQ.ID.NO.:583; antisense) 5′-ctag[GATATC]CGCAAAACCGTTTGCATATACTC-3′.

Nucleotides in lower caps are included as spacers just before therestriction sites between the endogenous TSHR and G protein. The senseand anti-sense primers included the restriction sites for XbaI andEcorV, respectively.

PCR was then utilized to secure the respective receptor sequences forfusion within the Gsα universal vector disclosed above, using thefollowing protocol for each: 100 ng cDNA for TSHR(A623K) was added toseparate tubes containing 2 μL of each primer (sense and anti-sense), 3μL of 10 mM dNTPs, 10 μL of 10×TaqPlus™ Precision buffer, 1 μL ofTaqPlus™ Precision polymerase (Stratagene: #600211), and 80 μL of water.Reaction temperatures and cycle times for TSHR were as follows: theinitial denaturing step was done at 94° C. for five minutes, and a cycleof 94° C. for 30 seconds; 55° C. for 30 seconds; 72° C. for two minutes.A final extension time was done at 72° C. for ten minutes. PCR productfor was run on a 1% agarose gel and then purified (data not shown). Thepurified product was digested with XbaI and EcorV (New England Biolabs)and the desired inserts isolated, purified and ligated into the Gsuniversal vector at the respective restriction site. The positive cloneswere isolated following transformation and determined by restrictionenzyme digest; expression using Hek-293 cells was accomplished followingthe protocol set forth infra. Each positive clone for TSHR: Gs-FusionProtein was sequenced and made available for the direct identificationof candidate compounds. (See, SEQ. ID. NO.:588 for nucleic acid sequenceand SEQ. ID. NO.:589 for amino acid sequence).

Location of non-endogenous version of TSHR(A623K) is located upstreamfrom the rat G protein Gsox (i.e., from nucleotide 1 through 2,292; see,SEQ. ID. NO.:586 and amino acid residue 1 through 764; see, SEQ. ID.NO.:587). TSHR(A623K) can be linked directly to the G protein, or therecan be spacer residues between the two. With respect to TSHR, 24 aminoacid residues (an equivalent of 72 nucleotides) were placed in betweenthe non-endogenous GPCR and the start codon for the G protein Gsu.Therefore, the Gs protein is located at nucleotide 2,365 through 3,549(see, SEQ. ID. NO.:586) and at amino acid residue 789 through 1,183(see, SEQ. ID. NO.:587). Those skilled in the art are credited with theability to select techniques for constructing a GPCR Fusion Proteinwhere the G protein is fused to the 3′ end of the GPCR of interest.

GPCR Fusion Protein was analyzed (to stabilize the GPCR while screeningfor candidate compounds, as shown in Example 6) and verified to beconstitutively active utilizing the protocol found in Example 4(2). InFIG. 5, TSHR(A623K)-Gαs:Fusion Protein evidenced about an 87% increasein cAMP when compared to the control vector (pCMV).

2. GPR24-Giα Fusion Protein

GPR24-Giα Fusion Protein construct was then made as follows: primerswere designed for both endogenous, constitutively activated andnon-endogenous, constitutively activated GPR24 were as follows:

(SEQ.ID.NO.:584; sense)     5′-GTGAAGCTTGCCCGGGCAGGATGGACCTGG-3′(SEQ.ID.NO.:585; antisense) 5′-ATCTAGAGGTGCCTTTGCTTTCTG-3′.

The sense and anti-sense primers included the restriction sites for KB4and XbaI, respectively.

PCR was then utilized to secure the respective receptor sequences forfusion within the Gict universal vector disclosed above, using thefollowing protocol for each: 100 ng cDNA for GPR24 was added to separatetubes containing 2 μL of each primer (sense and anti-sense), 3 μL of 10mM dNTPs, 10 μL of 10×TaqPlus™ Precision buffer, 1 μL of TaqPlus™Precision polymerase (Stratagene: #600211), and 80 μL of water. Reactiontemperatures and cycle times for GPR24 were as follows: the initialdenaturing step was done it 94° C. for five minutes, and a cycle of 94°C. for 30 seconds; 55° C. for 30 seconds; 72° C. for two minutes. Afinal extension time was done at 72° C. for ten minutes. PCR product forwas run on a 1% agarose gel and then purified (data not shown). Thepurified product was digested with KB4 and XbaI (New England Biolabs)and the desired inserts will be isolated, purified and ligated into theGi universal vector at the respective restriction site. The positiveclones was isolated following transformation and determined byrestriction enzyme digest; expression using Hek-293 cells wasaccomplished following the protocol set forth infra. Each positive clonefor GPR24: Gi-Fusion Protein was sequenced and made available for thedirect identification of candidate compounds. (See, SEQ. ID. NO.:590 fornucleic acid sequence and SEQ. ID. NO.:591 for amino acid sequence).

Endogenous version of GPR24 was fused upstream from the G protein Gi andis located at nucleotide 1 through 1,059 (see, SEE. ID. NO.:588) andamino acid residue 1 through 353 (see, SEQ. ID. NO.:589). With respectto GPR24, 2 amino acid residues (an equivalent of 6 nucleotides) wereplaced in between the endogenous (or non-endogenous) GPCR and the startcodon for the G protein Giα. Therefore, the Gi protein is located atnucleotide 1,066 through 2,133 (see, SEQ. ID. NO.:588) and at amino acidresidue 356 through 711 (see, SEQ. ID. NO.:589). Those skilled in theart are credited with the ability to select techniques for constructinga GPCR Fusion Protein where the G protein is fused to the 3′ end of theGPCR of interest.

Although it is indicated above that Gi coupled receptors, such as GPR24,can be used in conjunction with a co-transfection approach, this is inthe context of cAMP based assays and is predicated upon the effect of Gion cAMP levels. However, for other types of assays, such as a GTP basedassay, the co-transfection approach is not essential. Thus for assayssuch as a GTP based assay, the GPCR Fusion Protein approach is preferredsuch that, with respect to a GTP based assay for GPR24, the GPR24:GiFusion Protein would be preferred.

Example 6

Protocol: Direct Identification of Inverse Agonists and Agonists Using[³⁵S]GTPγS

Although Endogenous GPCRs may be utilized for the direct identificationof candidate compounds as, e.g., inverse agonists, for reasons that arenot altogether understood, intra-assay variation can become exacerbated.Preferably, then, a GPCR Fusion Protein, as disclosed above, can also beutilized with a non-endogenous, constitutively activated GPCR. We candetermine that when such a protein is used, intra-assay variationappears to be substantially stabilized, whereby an effectivesignal-to-noise ratio is obtained. This has the beneficial result ofallowing for a more robust identification of candidate compounds. Thus,it is preferred that for direct identification, a GPCR Fusion Protein beused and that when utilized, the following assay protocols be utilized.

1. Membrane Preparation

Membranes comprising the non-endogenous, constitutively active GPCRFusion Protein of interest and for use in the direct identification ofcandidate compounds as inverse agonists, agonists or partial agonistsare preferably prepared as follows:

a. Materials

“Membrane Scrape Buffer” is comprised of 20 mM HEPES and 10 mM EDTA, pH7.4; “Membrane Wash Buffer” is comprised of 20 mM HEPES and 0.1 mM EDTA,pH 7.4; “Binding Buffer” is comprised of 20 mM HEPES, 100 mM NaCl, and10 mM MgCl₂, pH 7.4.

b. Procedure

All materials will be kept on ice throughout the procedure. First, themedia is aspirated from a confluent monolayer of cells, followed byrinse with 10 ml cold PBS, followed by a aspiration. Thereafter, 5 ml ofMembrane Scrape Buffer will be added to scrape cells; this is followedby transfer of cellular extract into 50 ml centrifuge tubes (centrifugedat 20,000 rpm for 17 minutes at 4° C.). Thereafter, the supernatant isaspirated and the pellet is resuspended in 30 ml Membrane Wash Bufferfollowed by centrifugation at 20,000 rpm for 17 minutes at 4° C. Thesupernatant will then be aspirated and the pellet resuspended in BindingBuffer. This is then homogenized using a Brinkman Polytron™ homogenizer(15-20 second bursts until the all material is in suspension). This isreferred to herein as “Membrane Protein”.

2. Bradford Protein Assay

Following the homogenization, protein concentration of the membraneswill be determined using the Bradford Protein Assay (protein can bediluted to about 1.5 mg/ml, aliquoted and frozen (−80° C.) for lateruse; when frozen, protocol for use is as follows: on the day of theassay, frozen Membrane Protein is thawed at room temperature, followedby vortex and then homogenized with a Polytron at about 12×1,000 rpm forabout 5-10 seconds; it is noted that for multiple preparations, thehomogenizer should be thoroughly cleaned between homogenization ofdifferent preparations).

a. Materials

Binding Buffer (as per above); Bradford Dye Reagent; Bradford ProteinStandard are utilized, following manufacturer instructions (Biorad, cat.no. 500-0006).

b. Procedure

Duplicate tubes will be prepared, one including the membrane, and one asa control “blank”. Each contained 800 μL Binding Buffer. Thereafter, 10μL of Bradford Protein Standard (1 mg/ml) is added to each tube, and 10μL of membrane Protein is then added to just one tube (not the blank).Thereafter, 200 μL of Bradford Dye Reagent is added to each tube,followed by vortex of each. After five (5) minutes, the tubes will bere-vortexed and the material therein is transferred to cuvettes. Thecuvettes are then read using a CECIL 3041 spectrophotometer, atwavelength 595.

3. Direct Identification Assay

a. Materials

GDP Buffer consists of 37.5 ml Binding Buffer and 2 mg GDP (Sigma, cat.no. G-7127), followed by a series of dilutions in Binding Buffer toobtain 0.2 μM GDP (final concentration of GDP in each well was 0.1 μMGDP); each well comprising a candidate compound, will have a finalvolume of 200 μL consisting of 100 μL GDP Buffer (final concentration,0.1 μM GDP), 50 μL Membrane Protein in Binding Buffer, and 50 μL[³⁵S]GTPγS (0.6 nM) in Binding Buffer (2.5 μL [³⁵S]GTPγS per 10 mlBinding Buffer).

b. Procedure

Candidate compounds are preferably screened using a 96-well plate format(these can be frozen at −80° C.). Membrane Protein (or membranes withexpression vector excluding the GPCR Fusion Protein, as control) will behomogenized briefly until in suspension. Protein concentration is thendetermined using the Bradford Protein Assay set forth above. MembraneProtein (and control) is then diluted to 0.25 mg/ml in Binding Buffer(final assay concentration, 12.5 μg/well). Thereafter, 100 μL GDP Bufferwill be added to each well of a Wallac Scintistrip™ (Wallac). A 5 μLpin-tool is then used to transfer 5 μL of a candidate compound into suchwell (i.e., 5 μL in total assay volume of 200 μL is a 1:40 ratio suchthat the final screening concentration of the candidate compound is 10μM). Again, to avoid contamination, after each transfer step the pintool should be rinsed in three reservoirs comprising water (1×), ethanol(1×) and water (2×)—excess liquid should be shaken from the tool aftereach rinse and dried with paper and kimwipes. Thereafter, 50 μL ofMembrane Protein is added to each well (a control well comprisingmembranes without the GPCR Fusion Protein is also utilized), andpre-incubated for 5-10 minutes at room temperature. Thereafter, 50 μL of[³⁵S]GTPγS (0.6 nM) in Binding Buffer will be added to each well,followed by incubation on a shaker for 60 minutes at room temperature(again, in this example, plates were covered with foil). The assay isthen stopped by spinning of the plates at 4000 RPM for 15 minutes at 22°C. The plates will then be aspirated with an 8 channel manifold andsealed with plate covers. The plates are then read on a Wallacc 1450using setting “Prot. #37” (per manufacturer instructions).

Example 7

Protocol: Confirmation Assay

Using an independent assay approach to provide confirmation of adirectly identified candidate compound as set forth above, it ispreferred that a confirmation assay then be utilized. In this case, thepreferred confirmation assay is a cyclase-based assay.

A modified FLASH PLATE™ Adenylyl Cyclase kit (New England Nuclear; Cat.No. SMP004A) is preferably utilized for confirmation of candidatecompounds directly identified as inverse agonists and agonists tonon-endogenous, constitutively activated GPCR in accordance with thefollowing protocol.

Transfected cells will be harvested approximately three days aftertransfection. Membranes are prepared by homogenization of suspendedcells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl₂.Homogenization is performed on ice using a Brinkman POLYTRON™ forapproximately 10 seconds. The resulting homogenate will be centrifugedat 49,000×g for 15 minutes at 4° C. The resulting pellet is thenresuspended in buffer containing 20 mM IEPES, pH 7.4 and 0.1 mM EDTA,homogenized for 10 seconds, followed by centrifugation at 49,000×g for15 minutes at 4° C. The resulting pellet can be stored at −80° C. untilutilized. On the day of direct identification screening, the membranepellet is slowly thawed at room temperature, resuspended in buffercontaining 20 mM HEPES, pH 7.4 and 10 mM MgCl₂, to yield a final proteinconcentration of 0.60 mg/ml (the resuspended membranes are placed on iceuntil use).

cAMP standards and Detection Buffer (comprising 2 μCi of tracer [¹²⁵IcAMP (100 μl)] to 11 ml Detection Buffer) will be prepared andmaintained in accordance with the manufacturer's instructions. AssayBuffer will be prepared fresh for screening and contained 20 mM HEPES,pH 7.4, 10 mM MgCl₂, 20 mM phospocreatine (Sigma), 0.1 units/ml creatinephosphokinase (Sigma), 50 μM GTP (Sigma), and 0.2 mM ATP (Sigma); AssayBuffer can be stored on ice until utilized.

Candidate compounds identified as per above (if frozen, thawed at roomtemperature) will then be added, preferably, to 96-well plate wells (3μl/well; 12 μM final assay concentration), together with 40 μl MembraneProtein (30 μg/well) and 50 μl of Assay Buffer. This mixture is thenincubated for 30 minutes at room temperature, with gentle shaking.

Following the incubation, 100 μl of Detection Buffer is added to eachwell, followed by incubation for 2-24 hours. Plates are then counted ina Wallac MICROBETA™ plate reader using “Prot. #31” (as per manufacturerinstructions).

Example 8

Ligand-Based Confirmation Assay

Membranes will be prepared from transfected Hek-293 cells (see Example3) by homogenization in 20 mM HEPES and 10 mM EDTA, pH 7.4 andcentrifuged at 49,000×g for 15 min. The pellet will be resuspended in 20mM HEPES and 0.1 mM EDTA, pH 7.4, homogenized for 10 sec using Polytronhomogenizer (Brinkman) at 5000 rpm and centrifuged at 49,000×g for 15min. The final pellet will be resuspended in 20 mM HEPES and 10 mMMgCl₂, pH 7.4, homogenized for 10 sec using Polytron homogenizer(Brinkman) at 5000 rpm.

Ligand-based confirmation assays will be performed in triplicate 200 μlvolumes in 96 well plates. Assay buffer (20 mM HEPES and 10 mM MgCl₂, pH7.4) will be used to dilute membranes, tritiated inverse agonists and/oragonists and the receptor's endogenous ligand (used to definenon-specific binding). Final assay concentrations will consist of 1 nMof tritiated inverse agonist and/or agonist, 50 μg membrane protein(comprising the receptor) and 100 μm of endogenous ligand. Agonist assaywill be incubated for 1 hr at 37° C., while inverse agonist assays areincubated for 1 hr at room temperature. Assays will terminate by rapidfiltration onto Wallac Filtermat Type B with ice cold binding bufferusing Skatron cell harvester. The radioactivity will be determined in aWallac 1205 BetaPlate counter.

Again, this approach is used merely to understand the impact of thedirectly identified candidate compound on ligand binding. As those inthe art will appreciate, it is possible that the directly identifiedcandidate compounds may be allosteric modulators, (i.e., compounds thataffect the functional activity of the receptor but which do not inhibitthe endogenous ligand from binding to the receptor. Allostericmodulators include inverse agonists, partial agonists and agonists.

References cited throughout this patent document, including co-pendingand related patent applications, unless otherwise indicated, are fullyincorporated herein by reference. Modifications and extension of thedisclosed inventions that are within the purview of the skilled artisanare encompassed within the above disclosure and the claims that follow.

Although a variety of expression vectors are available to those in theart, for purposes of utilization for both the endogenous andnon-endogenous known GPCRs, it is most preferred that the vectorutilized be pCMV. This vector was deposited with the American TypeCulture Collection (ATCC) on Oct. 13, 1998 (10801 University Blvd.,Manassas, Va. 20110-2209 USA) under the provisions of the BudapestTreaty for the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure. The DNA was testedby the ATCC and determined to be. The ATCC has assigned the followingdeposit number to pCMV: ATCC #203351.

SEQUENCE LISTING The patent contains a lengthy “Sequence Listing”section. A copy of the “Sequence Listing” is available in electronicform from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=06806054B2). An electroniccopy of the “Sequence Listing” will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A method for directly identifying anon-endogenous compound as a compound having an activity selected fromthe group consisting of: inverse agonists, agonists, and partialagonists, to a non-endogenous, constitutively activated version of aknown G protein-coupled receptor, said receptor comprising atransmembrane-6 region and an intracellular region, comprising the stepsof: (a) selecting a non-endogenous version of a known GPCR; (b)confirming that the selected non-endogenous GPCR of step (a) isconstitutively active; (c) contacting a non-endogenous candidatecompound with the non-endogenous, constitutively activated GPCR of stepof (b); and (d) determining, by measurement of the compound efficacy atsaid contacted receptor, whether said non-endogenous compound is aninverse agonist, an agonist, or a partial agonist to said receptor ofstep (b); wherein said receptor of step (b) comprises the amino acidsequence of SEQ ID NO:449.
 2. The method of claim 1 further comprisingthe following step: assessing, by using an endogenous ligand basedassay, the impact of the non-endogenous compound of step (d) on thebinding of an endogenous ligand for the known GPCR version of thenon-endogenous version of said known GPCR of step (a) with said knownGPCR.
 3. The method of claim 2 wherein said non-endogenous compound isan allosteric modulator of said known GPCR.